Methods and compositions for synthetic biomarkers

ABSTRACT

The present disclosure encompasses embodiments of nucleic acids comprising genetic elements which are useful for the detection of diseased cells.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/US2020/026758, filed Apr. 4, 2020, which claims the benefit of U.S.Provisional Application 62/830,279, entitled “IMPROVED METHODS ANDCOMPOSITIONS FOR SYNTHETIC BIOMARKERS”, which was filed on Apr. 5, 2019and U.S. Provisional Application 62/955,925, entitled “IMPROVED METHODSAND COMPOSITIONS FOR SYNTHETIC BIOMARKERS”, which was filed on Dec. 31,2019, each of which is entirely incorporated herein by reference.

BACKGROUND

Cancer is an enormous global health problem. The World HealthOrganization estimates that in 2018 alone there were an estimated 18.1million new diagnoses of cancer and 9.6 million deaths due to cancer.The time at which cancer is detected, both prior to initial cancerdiagnosis and during tumor recurrence, is one of the most importantfactors affecting patient outcome since if detected early, currenttreatments are likely to be more effective. Unfortunately, the majorityof cancers are detected relatively late, leading to high mortalityrates. These rates are expected to double by 2030 unless more effectivedetection strategies and treatments are developed. To stem thetremendous loss of life due to this terrible disease, a broadlyapplicable tool capable of detecting cancer in its earliest stages isurgently needed.

Two current paradigms for improving cancer detection include thedevelopment of blood-based assays that detect endogenous cancerbiomarkers (e.g. protein, microRNA, circulating tumor DNA, circulatingtumor cells, etc.) that are shed or released into the bloodstream, andmolecular imaging assays that utilize biomarker-targeted imaging probesto better visualize tumors that are undetectable with conventionalanatomical imaging.

Blood assays are highly attractive as they facilitate affordable cancerscreening programs but often suffer from sensitivity and specificityissues due to low blood biomarker concentrations (Nagrath et al., (2007)Nature 450: 1235-1239), rapid in vivo and ex vivo biomarker degradation(Haun et al., (2011) Sci. Translational Med. 3: 71ra16), and highlyvariable background expression in non-malignant tissues (Diamandis E P(2010) J. National Cancer Inst. 102: 1462-1467). Using current clinicalbiomarker assays, it has been estimated that a tumor can grow for 10-12years and reach a spherical diameter greater than 2.5 cm beforeendogenous blood biomarker amounts reach sufficient levels to indicatedisease (Hori & Gambhir (2011) Sci. Translational Med. 3: 109ra116). Ofthe thousands of potential blood biomarkers reported, less than 1% areused in the clinic (7), and the implementation of new blood biomarkersinto the clinical setting is decreasing due to their lack of validatedspecificity and diagnostic value (Haun et al., (2011) Sci. TranslationalMed. 3: 71ra16; Kern S E (2012) Cancer Res. 72: 6097-6101). Overall,while enormous effort has been devoted to developing tools for detectingendogenous cancer blood biomarkers, there have been very few successes.Thus, new strategies and tools capable of sensitive and specific cancerdetection are urgently needed.

SUMMARY

In some aspects, the present disclosure provides for a methodcomprising: (a) administering to a subject a composition, wherein thecomposition induces expression of a biomarker in a diseased cellpreferentially over expression of the biomarker in non-diseased cells inthe subject such that a relative ratio of the biomarker expressed in thediseased cell over the non-diseased cells is greater than 1.0; (b)detecting the biomarker; and (c) using the biomarker detected in (b) todetermine that the subject has the diseased cell at an accuracy of atleast 70%.

In some aspects, the present disclosure provides for a method treating asubject having or suspected of having a disease, comprisingadministering to the subject a composition that induces expression of atherapeutically effective agent by a diseased cell associated with thedisease preferentially over expression of the therapeutically effectiveagent by non-diseased cells in the subject such that a relativeconcentration of the therapeutically effective agent expressed by thediseased cell over the non-diseased cells is greater than 1.0, whichtherapeutically effective agent treats the subject at a therapeuticefficacy of at least 10% as determined by a decrease in a cellpopulation of the diseased cells.

In some aspects, the present disclosure provides for a compositioncomprising a first nucleic acid sequence encoding a first polypeptide ornucleic acid biomarker and a second nucleic acid sequence encoding asecond polypeptide or second nucleic acid biomarker, wherein thecomposition is configured such that when the composition is in a cell:the second polypeptide or nucleic acid biomarker is expressed in anamount that reflects delivery of the first and the second nucleic acidsto the cell, and the first polypeptide or nucleic acid biomarker isexpressed differentially in a diseased cell versus a non-diseased cell.

In some aspects, the present disclosure provides for a method ofdetecting diseased cells in a subject, comprising administering acomposition to the subject, wherein the composition comprises: a firstnucleic acid sequence encoding a first polypeptide or nucleic acidbiomarker and a second nucleic acid sequence encoding a secondpolypeptide or second nucleic acid biomarker, wherein the composition isconfigured such that when the composition is in a cell: (i) the cellinduces expression of the first nucleic acid sequence in a diseased cellpreferentially over expression of the first nucleic acid sequence innon-diseased cells, wherein the first polypeptide is a detectablebiomarker or a therapeutic agent; and (ii) the cell induces equivalentexpression of the second nucleic acid sequence equally in diseased andin non-diseased cells and the second nucleic acid sequence yields thesecond polypeptide that is not the detectable biomarker or thetherapeutic agent, such that a level of expression of the secondpolypeptide provides a control for assessing the relative level of thenucleic acid sequences in the cell.

In some aspects, the present disclosure provides for a compositioncomprising a first nucleic acid sequence encoding a first polypeptideand a second nucleic acid sequence encoding a second polypeptide,wherein the composition is configured such that when the composition isin a cell: (i) the cell expresses the first nucleic acid sequence toyield the first polypeptide; (ii) the cell expresses the second nucleicacid sequence to yield the second polypeptide; and (iii) the firstpolypeptide and the second polypeptide expressed by the cell areconfigured to combine to form a heterodimer protein.

In some aspects, the present disclosure provides for a method ofdetecting or treating a diseased cell, comprising administering thecomposition above comprising a first nucleic acid sequence encoding afirst polypeptide and a second nucleic acid sequence encoding a secondpolypeptide, wherein the first and the second polypeptide areselectively transcribed or translated in the diseased cell.

In some aspects, the present disclosure provides for a compositioncomprising a non-naturally occurring recombinant genetic constructcomprising a sequence encoding a polypeptide or nucleic acid sequence,and wherein the sequence comprises a first promoter that selectivelydrives expression of the polypeptide or nucleic acid biomarker sequencein a plurality of different types of cells isolated from a subject whentransduced into the cells ex vivo.

In some aspects, the present disclosure provides for a method fordetecting a diseased or disordered cell ex-vivo, comprising deliveringex vivo a non-naturally occurring recombinant genetic construct to apopulation of cells isolated from a subject, wherein the non-naturallyoccurring recombinant genetic construct comprises: a sequence encoding apolypeptide or nucleic acid biomarker sequence, wherein the sequencecomprises a first promoter that selectively drives expression of thepolypeptide or nucleic acid biomarker sequence in a plurality ofdifferent types of cells isolated from a subject when transduced intothe cells.

In some aspects, the present disclosure provides for a compositioncomprising a vector, wherein the vector comprises a plurality ofdifferent promoters operably linked to a plurality of different nucleicacid sequences, wherein each the promoter drives expression of theplurality of nucleic acid sequences in a cell to yield a plurality ofpolypeptides or nucleic acid biomarker sequences, wherein levels ofindividual polypeptides or nucleic acid biomarker sequences of theplurality of nucleic acid sequences are indicative of a stage of adisease of the cell, or a tissue from which the cell originates.

In some aspects, the present disclosure provides for a method fordetecting a stage of disease, comprising administering to a subject acomposition comprising a vector, wherein the vector comprises aplurality of different promoters operably linked to a plurality ofdifferent nucleic acid sequences, wherein each the promoter drivesexpression of the plurality of nucleic acid sequences in a cell to yielda plurality of polypeptides or nucleic acid biomarker sequences, whereinlevels of individual polypeptides of the plurality of nucleic acidsequences are indicative of a stage of a disease of the cell, or atissue from which the cell originates.

In some aspects, the present disclosure provides for a compositioncomprising an engineered nucleic acid encoding an expressible reportergene that exhibits about 10% or less expression in normal cells versusdiseased cells when compared to a recombinant nucleic acid comprising areporter gene comprising a nucleic acid sequence of SEQ ID NO: 1 or SEQID NO: 2.

In some aspects, the present disclosure provides for a method comprisingadministering to a subject the composition comprising the engineerednucleic acid encoding an expressible reporter gene above.

In some aspects, the present disclosure provides for a composition thatexhibits about 10% or less expression in normal cells versus diseasedcells and comprises a recombinant nucleic acid comprising a nucleic acidsequence encoding a reporter gene that includes one or more miRNAbinding sequences in a 3′ untranslated region of the reporter gene.

In some aspects, the present disclosure provides for a method ofdetecting a diseased cell comprising administering to a subject thecomposition that exhibits about 10% or less expression in normal cellsversus diseased cells above.

In some aspects, the present disclosure provides for a compositionexhibiting significantly longer expression of synthetic biomarker versusplasmid DNA or minicircle DNA comprising a linear vector comprising adouble-stranded nucleic acid comprising a promoter operatively linked toa DNA sequence encoding a synthetic biomarker, wherein a forward and areverse strand of the double-stranded nucleic acid are covalently linkedon each of their terminal ends, wherein the promoter induces expressionof the synthetic biomarker in a diseased cell preferentially overexpression of the synthetic biomarker in a non-diseased cell such that arelative concentration of the synthetic biomarker expressed in thediseased cell over the non-diseased cell is greater than 1.0.

In some aspects, the present disclosure provides for a method ofidentifying a diseased cell, comprising administering to a subject thecomposition exhibiting significantly longer expression of syntheticbiomarker versus plasmid DNA or minicircle DNA above, and detecting thesynthetic biomarker, wherein the synthetic biomarker is expressed in adiseased cell preferentially over expression of the synthetic biomarkerin non-diseased cells in the subject such that a relative concentrationof the synthetic biomarker expressed in the diseased cell over thenon-diseased cells is greater than 1.0.

In some aspects, the present disclosure provides for a compositionexhibiting significantly longer expression of synthetic biomarker versusplasmid DNA or minicircle DNA comprising a linear vector comprising adouble-stranded nucleic acid comprising a promoter operatively linked toa DNA sequence encoding a therapeutically effective agent, wherein aforward and a reverse strand of the double-stranded nucleic acid arecovalently linked on each of their terminal ends, wherein the promoterinduces expression of the therapeutically effective agent in a diseasedcell preferentially over expression of the synthetic biomarker in anon-diseased cell such that a relative concentration of thetherapeutically effective agent expressed in the diseased cell over thenon-diseased cell is greater than 1.0.

In some aspects, the present disclosure provides for a method oftreating a diseased cell, comprising administering to a subject thecomposition above, and detecting the synthetic biomarker, wherein thesynthetic biomarker is expressed in a diseased cell preferentially overexpression of the synthetic biomarker in non-diseased cells in thesubject such that a relative concentration of the synthetic biomarkerexpressed in the diseased cell over the non-diseased cells is greaterthan 1.0.

In some aspects, the present disclosure provides for a compositioncomprising a non-viral vector expressing a synthetic biomarker, whereinthe synthetic biomarker exhibits about 10% or less expression in normalorgan cells versus diseased cells.

In some aspects, the present disclosure provides for an engineeredparticle that mimics one or many functions of a biological cell ormacrophage including inducing the expression of a biomarker in adiseased cell preferentially over expression of the biomarker innon-diseased cells such that the relative concentration ratio of thebiomarker expressed in the diseased cell over the non-diseased cells isgreater than 1.0.

In some aspects, the present disclosure provides for at least onevector, wherein the at least one vector comprises: a plurality ofdifferent promoters operably linked to a plurality of different nucleicacid sequences, wherein the promoters drive expression of the pluralityof nucleic acid sequences in a cell to yield a plurality of polypeptidesor nucleic acid biomarker sequences, wherein the promoters induceexpression of the plurality of polypeptides or nucleic acid biomarkersequences in a diseased cell preferentially over expression of theplurality of polypeptides or nucleic acid biomarker sequences innon-diseased cells in a subject such that a relative ratio of theplurality of polypeptides or nucleic acid biomarker sequences expressedin the diseased cell over the non-diseased cells is greater than 1.0.

In some aspects, the present disclosure provides for a method fordetecting a disease in a subject, comprising: administering to a subjecta composition comprising the at least one vector comprising a pluralityof different promoters operably linked to a plurality of differentnucleic acid sequences above; detecting the plurality of polypeptides ornucleic acid biomarker sequences to obtain an expression profile; anddetecting the diseased cell based expression profile, thereby detectingthe disease.

In some aspects, the present disclosure provides for methods fordetecting a subject's disease or absence thereof, comprising contactingone or more cells of said subject with a genetic construct ex-vivo,wherein: said genetic construct comprises a disease-activated promoteroperably linked to a barcode molecule and said disease-activatedpromoter drives expression of said barcode molecule in a cell affectedby said disease; quantifying an expression level of said barcodemolecule; and detecting said disease or absence thereof based on saidexpression level.

By ascribing an exclusive label to a unique member within a largergroup, barcodes afford the opportunity to identify and quantify thatmember (e.g. expression of a reporter under the control of a particularcancer specific promoter) within the context of a larger and morecomplex mixture of many members (e.g. multiple promoter-reporterconstructs expressed within the same cell), as well as offering theopportunity to isolate a single member from the complex mixture. Forinstance, in the case of barcodes based on nucleic acids, hybridizationof barcodes based on base pairing complementarity may be used to captureand isolate or otherwise reduce the complexity of a mixture by saidcapture event. For barcodes based on peptides, unique features includingimmunocapture or interactions of ligands and receptors may be used tocapture and isolate or otherwise reduce the complexity of a mixture bysaid capture event.

In some aspects, the present disclosure provides for methods forgenerating a profile of a subject's disease, comprising contacting oneor more cells of said subject with a plurality of genetic constructs,wherein: said plurality of genetic constructs comprises a plurality ofdisease-activated promoters respectively operably linked to a pluralityof barcode molecules and said disease-activated promoter drivesexpression of said corresponding barcode molecule in a cell affected bysaid disease; and quantifying expression levels of said plurality ofbarcode molecules to generate said profile.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (denoted “FIGURE” or “FIG.”) of which:

FIG. 1 schematically illustrates a blood-based tumor-activatableminicircle (MC) approach for cancer detection. (A) Tumor-activatable MCsdriven by a tumor-specific promoter and encoding a secretable reporterprotein are complexed with a non-targeted transfection agent (TA). Thesenanocomplexes are delivered systemically (via tail-vein). (B) MCstransfect many tissues, but reporter protein production occursnear-exclusively within tumor cells and the expressed reporter issecreted into the bloodstream (BS). Minimal protein expression shouldoccur in tumor-free subjects due to promoter leakiness. (C) Collectionof blood and detection of the secreted reporter in plasma enablesdifferentiation between tumor-bearing (reporter-positive) and tumor-free(reporter-negative) subjects.

FIGS. 2A-2B illustrate the design and construction of tumor-activatablevectors. FIG. 2A illustrates vector maps of both Survivin promoter(pSurv)-driven parental plasmids (PP; top) and MCs (bottom). Theseconstructs encoded the reporter protein secreted embryonic alkalinephosphatase (SEAP). The PP and MC have the identical transcription unit(pSurv-SEAP-WPRE-polyA) but the MC lacks the prokaryotic backbone (lightgrey). WPRE (Woodchuck Hepatitis virus posttranscriptional regulatoryelement (WPRE). FIG. 2B illustrates agarose gel electrophoresiscon-firming the ability to generate both PP (7.9 kb) and MC (4.1 kb).

FIG. 3 is a schematic map of minicircle vector constructMC-pSurv-SEAP-WPRE-SV40PolyA-pause.

FIG. 4 is a schematic map of minicircle vector constructMC-pSurv-Luc2-WPRE-SV40PolyA-pause.

FIG. 5 is a graph illustrating a comparison of tumor-specific plasmids(PP-SEAP) and minicircles (MC-SEAP) in MeWo human melanoma cancer cells.

FIG. 6 is a graph illustrating a comparison of tumor-specific plasmids(PP-SEAP) and minicircles (MC-SEAP) in SK-MEL-28 human melanoma cancercells.

FIG. 7 is a SEAP assay standard curve for blood-based cancer detectionafter systemic administration of tumor-specific SEAP Minicircles.Standard curve analysis of the SEAP assay revealed that RLU values aboveapproximately 10⁴ were within the linear region of detectable SEAPlevels in plasma.

FIG. 8 is a graph illustrating that intratumoral administration oftumor-activatable MCs leads to detectable blood reporter activity. Nudemice bearing subcutaneous human melanoma xenografts were intratumorally(I.T.) administered tumor-activatable MCs expressing SEAP (n=4; MC I.T.)or 5% glucose (n=3; Mock). A group of control mice also receivedintramuscular (I.M.) injections of MCs (n=3; MC I.M.). Plasma SEAPmeasurements before and for up to 2 weeks following MC administrationrevealed that only MC I.T. mice had elevated SEAP levels from days 3 to14 (*p<0.05; **p<0.01; ***p<0.001). Data is expressed as mean±SD.

FIG. 9 illustrates representative mouse blood SEAP activity aftersystemic administration of tumor-specific SEAP minicircles.

FIG. 10 is a graph illustrating blood-based cancer detection aftersystemic administration of tumor-specific SEAP minicircles.Significantly higher SEAP activity was detected in blood samples fromtumor-bearing mice than control mice from days 3 to 14 post-injection ofpSurvivin-SEAP MCs (p<0.05). No significant differences were notedbetween control mice receiving MC or 5% glucose. Error bars representSD.

FIG. 11 is a series of digital images illustrating molecular-geneticimaging cancer detection 3 days after systemic administration oftumor-specific FLUC minicircles.

FIG. 12 is a graph illustrating molecular-genetic imaging cancerdetection after systemic administration of tumor-specific FLUCminicircles.

FIG. 13 illustrates the nucleic acid sequence of minicircleMC-pSurv-SEAP-WPRE-pA.

FIG. 14 illustrates the nucleic acid sequence of minicircleMC-pSurv-Luc2-WPRE-pA.

FIG. 15 is a graph illustrating a comparison of the transfections of theconstructs of the disclosure in cultured cancer cells. Transfection ofequal mass of MC (n=3) and PP (n=3) using equal volumes of transfectionagent into MeWo human melanoma cells lead to significantly higher SEAPconcentration in medium with MCs from day 3 to day 8 (**p<0.01;***p<0.001). Data is expressed as mean±SD.

FIGS. 16A-16D illustrate the systemic delivery of tumor-activatable MCsallowing identification of tumor-bearing subjects. FIGS. 16A-16Cillustrate human melanoma tumor development following intravenous celladministration in nude mice (n=7) monitored using bioluminescenceimaging (BLI) (left images). Representative BLI images showed tumorgrowth primarily within the lung and individual mice had a wide range oftumor burden within 3 days prior to MC administration. The BLI scales inFIGS. 16A and 16B are the same, but that of FIG. 16C is one order ofmagnitude lower. Tumor-activatable MCs were administered systemically,and SEAP levels were measured before (Day 0) and up to 14 days followingadministration (right graphs). Varying SEAP concentrations were detectedin tumor-bearing mice over the 14-day period. FIG. 16D illustrateshealthy (tumor-free) mice that received either MC (Control+MC; n=7) or5% glucose carrier only (Control-MC; n=5). No statistically significantdifferences in plasma SEAP levels were detected between these twogroups. Importantly, across all mice regardless of tumor burden,significantly higher plasma SEAP concentration was detected intumor-bearing mice receiving MC between days 3 to 14 compared to bothcontrol groups (#*p<0.05; ##**p<0.01). Data is expressed as mean±SEM.

FIGS. 17A-17C illustrate that tumor-activatable MCs can robustlyidentify tumor-bearing subjects and measure tumor burden. FIG. 17A: Areaunder the curve (AUC) analysis of plasma SEAP measurements over 2 weeksrevealed significant differences between tumor-bearing mice receivingMCs (n=7) compared to both healthy mice receiving MCs (n=7) or 5%glucose (n=5) (*p<0.05; **p<0.01). Data is expressed as meant SD. FIG.17B: ROC (receiver operating characteristic curve) analysis revealed asignificant ability of the tumor-activatable MC system to differentiatetumor-bearing from healthy subjects by measuring and computing plasmaSEAP AUC. FIG. 17C: Correlational analysis of SEAP AUC measurements andlung tumor burden (as measured by BLI lung average radiance). Across 6mice a significant positive correlation was noted between these twomeasures, showing the ability of our tool to assess tumor burdenprovided that the tumor is in one location. One mouse was removed fromanalysis (square symbol) since this mouse had tumors in both the lungsand multiple metastatic foci outside the lungs (BLI measurement wastaken from just within lung explaining overall low BLI signal in thismouse). This mouse had a higher SEAP AUC level than would be expectedbased on its lung tumor burden.

FIGS. 18A-18D illustrate comparison of promoter activities in vivo inhealthy (tumor-free) mice. Mice were systemically administered plasmids(30 μg; PGL4.2 back-bone; complexed with PEI (N/P=6)) expressing thebioluminescence imaging (BLI) reporter gene codon-optimized fireflyluciferase (Luc2) driven by pCMV (n=3), pSury (n=5), or pPEG (n=3).Mock-injected mice received 5% glucose (n=3). Each mouse was alsoco-injected with a plasmid expressing the BLI reporter gene humanizedRenilla luciferase (hRluc) driven by pCMV to assess transfectionefficiency (3 μg; 10-fold less than Luc2 plasmid mass). FIG. 18Aillustrates representative BLI images 48 hours post-injection. Imagescale for the pCMV mouse is 2 orders of magnitude higher than all othermice. BLI signal, primarily in the lungs, was seen in all mice receivingLuc2 plasmid. FIG. 18B illustrates region-of-interest analysis over theentire mouse performed on BLI images, revealing significantly higher(*p<0.05; about 100-fold) BLI signal in mice receiving pCMV-Luc2plasmids compared to all other mice (*p<0.05). A significantly higher(*p<0.05) BLI signal was also observed in pPEG mice compared tomock-injected mice. Although qualitatively higher BLI signal was notablein pSury mice compared to mock-injected mice, quantitative measures onlyrevealed a trend (p=0.16) towards higher BLI signal. Thus, in this mousestrain, Luc2 expression in normal tissues was lowest with thetumor-specific pSurv. FIG. 18C illustrates that 48 h after plasmidinjection, ex vivo analysis of Luc2 activity across numerous tissuesrevealed significantly higher (*p<0.05) expression with pCMV compared toall other groups. With pPEG, significantly higher (*p<0.05) Luc2activity was found in the heart, lung and spleen compared tomock-injected animals. With pSurv, significantly higher (*p<0.05) Luc2activity was in the spleen and a trend (p=0.13) towards higher activityin the lung. FIG. 18D illustrates that the only tissue showing higherhRluc activity above background was the lung (values presented arenormalized to average background values from mock-injected mice). Due tothis, Luc2 values determined from both imaging (FIG. 18B) and ex vivotissue analysis (FIG. 18C) are not normalized by hRluc values. Nosignificant differences in hRluc values within the lungs were seenacross the 3 promoter mouse groups. Thus, differences Luc2 measurementsacross the 3 groups are unlikely to be related to differences intransfection efficiency but to differences in promoter activity. Data isexpressed as mean±SD.

FIGS. 19A-19C illustrate a comparison of tumor-specific promoteractivities in primary human fibroblasts and human cancer cell lines.Primary human fibroblasts, MDA-MB-231 cells (human breast cancer) andMeWo cells (human melanoma) were transfected with pPEG- or pSurv-drivenplasmids (1 μg) expressing Luc2 and co-transfected with a promoterlessplasmid expressing hRluc (50 ng) to normalize for transfectionefficiency. No differences in Rluc transfection efficiency were noted inany of the 3 cell types. pPEG-driven plasmids led to significantlyhigher Luc2 activity in fibroblasts than pSury (*p<0.05). pSurv-drivenplasmids led to significantly higher Luc2 activity in MeWo cells(***p<0.001) and equivalent activity in MDA-MB-231 cells. Data isexpressed as mean±SD.

FIG. 20 illustrates a comparison of tumor-activatable PPs and MCs incultured SK-MEL-28 melanoma cells. SK-MEL-28 human melanoma cells weretransfected with equal masses of tumor-activatable MC (n=3) and PP (n=3)and equal volumes of transfection agent PEI. Significantly higher SEAPactivity was observed in medium of cells transfected with MCs from 10day 2 to day 7 (**p<0.01; ***p<0.001). Data is expressed as mean±SD.

FIGS. 21A and 21B illustrate a comparison of trans-gene expressionbetween MCs and PPs driven by a strong constitutive promoter in healthy(tumor-free) mice. FIG. 21A illustrates mice that received systemicadministration of either MCs (n=4) or PPs (n=5) expressing hRluc drivenby the strong constitutive EF1 promoter after complexation with PEI (40μg; N/P=8). BLI imaging was performed on days 1, 2, 3, 5 and 7 using thesubstrate coelenterazine. Representative images show higher BLI signalin MC-administered mice at all time points examined. Signal from a mousereceiving a 5% glucose injection is shown for comparison (signal inliver is from oxidized coelenterazine). FIG. 21B illustrates aregion-of-interest analysis over the lung region showing a significantlyhigher BLI signal in MC versus PP mice on days 1, 2, and 5 (*p<0.05;**p<0.01). Data is expressed as mean±SD.

FIG. 22A illustrates a standard curve analysis of plasma SEAP assay.Triplicate samples were measured at 10-fold dilutions of SEAP in 25 IAof plasma. SEAP activity was linear over 5 orders of magnitude andshowed a detection limit of approximately 3×10⁻⁷ μg (0.3 pg) in 25 μl ofplasma. FIG. 22B illustrates SEAP measures over the entire linear rangethat were reproducible with coefficient of variance (% CV) measures lessthan 4%.

FIG. 23 illustrates tumor burden before and after MC Administration.Bioluminescence (BLI) images (left) of two representative mice (top andbottom) prior to and two weeks following MC administration andcorresponding ex vivo images (right) of lungs at time of sacrifice (2weeks after MC administration). Values below each BLI image representaverage radiance in regions of interest drawn over the lungs. There is adifference in image scales for the two mice. Indicating continual tumorgrowth, both mice showed an approximate 4.5-fold increase in BLI signalover the 2-week period following MC administration. At sacrifice, tumorswithin the lungs were melanotic and multiple tumor foci throughout thelungs were observed in both mice (white arrows). Based on BLI signalchanges, total tumor burden at the time of MC administration (two weeksprior to sacrifice) would have been approximately 4.5-fold less thanthat seen in the ex vivo images presented here.

FIG. 24 illustrates the results of the experiment of Example 11, dopingFLuc expressing cells into normal PBMCs, demonstrating that the limit ofdetection for such a detection method is at least 3-10 diseased cellsper 5 million normal PBMCs.

FIG. 25 illustrates the results of the experiment of Example 12,cancer-activated DNA constructs differentiate tumor-bearing and healthymice: following intravenous administration of surviving-SEAP DNAnanoplasmids, whole blood was collected by submandibular bleeds andprocessed into plasma. SEAP assays were performed on 20 μl aliquots.Cohort size are n=5.

FIGS. 26A-26F illustrate in vivo efficacy and in vitro cytotoxicity ofpolymer/DNA complex. FIG. 26A, shows an experiment where 40 μg of DNAencoding CMV-Luciferase was formulated in the polymeric formulations orwas complexed with JetPEI followed by intravenous administration intoBalb/C mice, D-luciferin was injected into the animals four days aftertransfection, after which the animals were sacrificed and lungs wereharvested for ex vivo BLI analysis. For cytotoxicity assessments (FIGS.26B, 26C, 26D, 26E, and 26F), polyplexes containing 250 ng of CMV-LucDNA were added into each well of a 96-well plate with 10,000 cellsplated per well the day before transfection. Each formulation was testedwith 3 replicates. 48 hours later, cell morphology was recorded bymicroscope and cell viability was measured during the MTT assay; FIG.26B shows blank cells; FIG. 26C shows in vivo delivery of construct withJetPEI; FIG. 26D shows delivery of DNA with high-molecular-weight,amine-terminated poly(β-amino ester) C32-122; FIG. 26E shows delivery ofDNA with high-molecular-weight, amine-terminated poly(β-amino ester)C32-145; and FIG. 26F shows cell viability results by MTT assays.

FIG. 27 illustrates that protamine condenses DNA polyplex size. 62.5 μgof DNA was condensed by thoroughly mixing with 130 μg of protamine at1:1 v/v in 50 mM Sodium acetate buffer (pH=5.0). The DNA/protaminecomplex was then diluted to 1.5 mL with 50 mM Sodium acetate buffer(pH=5.0). The Protamine: DNA LNP was assembled on a NanoAssemblr(Precision NanoSystems) with a total flow rate of 12 mL/min. Theas-prepared particles were dialyzed against 1×PBS for at least 18 h,after which the size was determined by a Zetasizer.

FIG. 28 illustrates an example of using luciferase to determinebiodistribution of delivery formulations. A) In vivo bioluminescenceimaging (BLI) of a mouse administered a tail vein injection with 40 mgof a CMV luciferase vector that had been formulated in JetPEI. Four dayspost administration, the mouse was anesthetized, administeredD-luciferin substrate and imaged now on an AMI-HT (Spectral InstrumentsImaging). B) After in vivo imaging, the mouse was sacrificed, and organsharvested for ex vivo BLI.

FIGS. 29A and 29B illustrates sensitivity and specificity ofAd-Survivin-FLuc in an ex vivo assay in canine PBMCs and cells derivedfrom canine tumors. Naïve, untransduced cells derived from varioussubtypes of canine malignancies including osteosarcoma, melanoma andhemangiosarcoma were spiked into 5e5 canine PBMCs and then transducedwith 0.3 MOI of Ad-Survivin-FLuc. A) Analysis demonstrates single celldetection of A17 osteosarcoma cells or B) robustness of detection acrosscells derived from multiple tumor types.

FIGS. 30A-C illustrate sensitivity and specificity of Ad-survivin-FLucin an ex vivo assay. A) H1299 cells, engineered to constitutivelyexpress the firefly luciferase protein, were spiked into 5e6 humannormal PBMCs and then the entirety of the sample was processed andanalyzed for luciferase expression. B) naïve H1299 cells, which had notbeen transduced, were spiked into the human PBMCs and then transducedwith a recombinant adenovirus with an expression cassette of humansurvivin promoter driving the expression of firefly luciferase(Ad-Survivin-FLuc). After growth for 48 hours, the sample was processedand analyzed for luciferase expression. C) Samples with only human PBMCswith transduced with either Ad-Survivin-FLuc or Ad-CMV-FLuc, the latter,under control of a strong constitutive promoter. Following a 2-dayincubation, luminescence assays were used to quantify FLuc expression.

FIG. 31 shows that Ad-survivin-FLuc distinguishes cancer vs normal in anex vivo assay on human PBMCs. Commercially available samples of humanPBMCs from normal healthy volunteers and cancer patients wereenumerated, and then equivalent numbers of cells were transduced atconsistent MOI with Ad-survivin-Fluc and then the samples split intotriplicate. Following incubation for three days, the cells were lysedand analyzed for luciferase activity. Data is calculated as the averageand Std Dev of the triplicate sample measurements. P-values werecalculated by Students T-test relative to normal PBMCs.

FIGS. 32A and 32B show a diagnostic performance of survivin-activatedluciferase expression in discerning healthy canine individuals andcanine lymphoma cancer patients. (A) Comparison of the fold-change inluminescence expression of healthy canine individuals (n=31) and caninelymphoma cancer patients (n=17). (B) Diagnosis predictive capacity ofsurvivin-activated luciferase activity to distinguish canine lymphomacancer subjects and healthy canine subjects.

FIGS. 33A, 33B, 33C, 33D, 33E, and 33F illustrate the activation ofvarious promoter-reporter constructs in particular cell lines of varyingtissue origin, where the gene labels denotes the source of promotersused in the construct. FIG. 33A shows various promoter-reporterconstructs in cell lines of liver origin (e.g. HepG2 and Hep3B),demonstrating that CXCR4, TRIP13, MCM10, COL10A1, BIRC5, and BIRC5-501are particularly activated in liver cancers. FIG. 33B shows activationof various promoter-reporter constructs in immortalized cell lines ofovarian origin (e.g. SKOV3 and OVCAR), demonstrating that COL10A1,MMP13, UBE2C, MUC1, CEP55, CEACAM5, KIF20A, FAM111B, and CST1 areparticularly activated in ovarian cancers. FIG. 33C shows activation ofvarious promoter-reporter constructs in immortalized cell lines ofpancreatic origin (e.g. ASPC1, BXPC3, and PANC1), demonstrating thatBIRC5, ABCC4, MMP13, CXCR4, UBE2C, MUC1, CDKN3, MCM10, CDC20, CEP55,CEACAM5, KIF20A, CST1, and FAM111B are particularly activated inpancreatic cancers. FIG. 33D shows activation of variouspromoter-reporter constructs in a cell line of breast origin (e.g.MDA-MB-231), demonstrating that BIRC5, MCM10, MMP1, DTL, CEP55, KIF4A,RGS13, KIF20A, UBE2T, CENPF, CST1, TOP2A, FAM111B, and MMP13 areparticularly activated in breast cancers. FIG. 33E shows activation ofvarious promoter-reporter constructs in cell lines of lung origin (e.g.A549, H460, and H1299), demonstrating that MCM10, AFP, MMP1, CEP55,CEACAM5, RGS13, KIF20A, CST1, FAM111B, and MMP13 are particularlyactivated in lung cancers. FIG. 33F shows a comparison of the samepromoter-reporter constructs as 33E but in non-transformed breast cancerlines, demonstrating that genes other than MMP1 that are shown activatedin 33E may be particularly useful for distinguishing breast cancer fromnormal tissue.

FIGS. 34A, 34B, and 34C illustrate the activation of a panel ofpromoter-reporter constructs in melanoma, osteoscarcoma, andhemangiosarcoma cancer cell lines, where the gene labels denotepromoters used in the construct. FIG. 34A shows activation of members ofthe panel in cell lines of melanoma origin (e.g. M2, M3, M4, M5, andCMGD), demonstrating that BIRC5, BIRC5-501, CXCR4, UBE2C, TRIP13, CDKN3,MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are particularlyactivated in melanoma cancer. FIG. 34B shows activation of members ofthe panel in cell lines of osteosarcoma origin (e.g. OS17, OS29, OS40,and OS484), demonstrating that BIRC5, BIRC5-501, CXCR4, UBE2C, TRIP13,CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are particularlyactivated in osteosarcoma. FIG. 34C shows activation of members of thepanel in cell lines of hemangiosarcoma origin, showing that BIRC5,BIRC5-501, MMP13, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP,CEP55, KIF20A, and cBIRC5 are particularly activated in hemangiosarcomacancer.

FIGS. 35A, 35B, and 35C show design of a multiplex detection assay usingmultiple different cancer-specific promoters and linked barcodes. 35Ashows design of the multiplex constructs, wherein various cancerspecific promoters (designated non-descriptively as Px, Py, and Pz) areused to drive expression of orthogonal reporters created by fusion of asignal peptide to luciferase with an intervening nucleic acid barcodesequence unique to the promoter being used to drive the construct(Barcodes A, B, and C for promoters Px, Py, and Pz, respectively). FIG.35B demonstrates relative expression of each promoter construct whenindividually transfected in equimolar amounts into H1299 cells and FIG.35C shows relative expression of each promoter construct whencombinatorially transfected in equimolar amounts into H1299 cells,demonstrating that co-transfection of multiple reporter-promoterconstructs into the same cells does not appreciably alter the expressionpattern of a given promoter in a given cell line, indicating that themultiplex format is a viable format for generating “profiles” ofpromoter activation in single cell types.

FIGS. 36A, 36B, and 36C show design of a multiplex detection assay usingmultiple different peptide epitopes to detect reporters driven fromseparate promoters. 36A shows design of the multiplex constructs,wherein different copies of the CMV promoter drive expression oforthogonal reporters created by fusion of a signal peptide to luciferasewith an intervening epitope peptide (e.g. FLAG, HA, V5, or HSV peptideepitopes) barcode that is unique to the promoter being used to drive theconstruct (36A shows CMV promoter being used, but ultimately multipledistinct promoters such as the Px, Py, Pz, etc of FIG. 35 isenvisioned). FIG. 36B demonstrates how the multiple epitope barcodes canbe used with capture antibodies specific for the epitopes (e.g.anti-FLAG, anti-HA, anti-V5, or anti-HSV) to separate out the secretedreporter constructs to obtain independent measures of the activities foreach promoter. FIG. 36C shows an example using theFLAG/HA/V5/HSV-barcoded luciferase constructs co-transfected into cells,demonstrating that luciferase constructs tagged with each peptideepitope can be separated and used to independently read out promoteractivation in a same cell.

FIGS. 37A, 37B, and 37C show a design of a reporter-promoter constructdesigned to use an off-the-shelf lateral flow assay (e.g. a pregnancyhCG lateral flow immunoassay) to detect activation of apromoter-reporter construct in a cancer cell line, along withcorresponding performance data. In this design, a cancer specificpromoter (Px, in this case represented by the survivin promoter) is usedto drive expression of a secretion-signal modified luciferase also fusedto a human chorionic gonadotropin (hCG) epitope. FIG. 37B shows viatransfection of various related constructs into H1299 cells that the hCGtag does not appreciably disrupt expression of luciferase from thesurvivin promoter. FIG. 37C shows that the supernatant from thetransfected cells can be loaded onto a commercial lateral-flowimmunoassay strip for hCG and that the lateral flow immunoassay candetect the hCG-tagged luciferase, showing the utility of using exitingepitope immunoassays to read out the expression of promoter-reporterconstructs where the reporter is tagged with an epitope having ahigh-confidence off-the-shelf assay.

FIG. 38 shows an example nanoplasmid-based promoter construct asdescribed herein. The sequence of this construct is outlined in SEQ IDNO: 5, and involves a mini-R6K origin, an RNA-out selectable marker, asurvivin promoter, SEAP as a reporter, and a WPRE element.

DETAILED DESCRIPTION OF THE INVENTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, toxicology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. In this disclosure, “comprises,”“comprising,” “containing” and “having” and the like can have themeaning ascribed to them in U.S. patent law and can mean “includes,”“including,” and the like; “consisting essentially of or “consistsessentially” or the like, when applied to methods and compositionsencompassed by the present disclosure refers to compositions like thosedisclosed herein, but which may contain additional structural groups,composition components or method steps (or analogs or derivativesthereof as discussed above). Such additional structural groups,composition components or method steps, etc., however, do not materiallyaffect the basic and novel characteristic(s) of the compositions ormethods, compared to those of the corresponding compositions or methodsdisclosed herein.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

Definitions

The term “subject” can include human or non-human animals. Thus, themethods and compositions described herein are applicable to both humanand veterinary disease and animal models. Preferred subjects are“patients,” i.e., living humans that are receiving medical care for adisease or condition. This includes persons with no defined illness whoare being investigated for signs of pathology. Also included are personssuspected of possessing or being at-risk for a defined illness.

The term “gene,” as used herein refers to all regulatory and codingsequences contiguously associated with a single hereditary unit with agenetic function. Genes can include non-coding sequences that modulatethe genetic function that include, but are not limited to, those thatspecify polyadenylation, transcriptional regulation, DNA conformation,chromatin conformation, extent and position of base methylation andbinding sites of proteins that control all of these. Genes encodingproteins are comprised of “exons” (coding sequences), which may beinterrupted by “introns” (non-coding sequences). In some instances,complexes of a plurality of protein or nucleic acids or other molecules,or of any two of the above, may be required for a gene's function. Onthe other hand, a gene's genetic function may require only RNAexpression or protein production or may only require binding of proteinsand/or nucleic acids without associated expression. In certain cases,genes adjacent to one another may share sequence in such a way that onegene will overlap the other. A gene can be found within the genome of anorganism, in an artificial chromosome, in a plasmid, in any other sortof vector, or as a separate isolated entity.

The terms “episomally replicating vector” or “episomal vector” as usedherein refer to a vector which is typically not integrated into thegenome of the host cell but exists in parallel. An episomallyreplicating vector may be replicated during the cell cycle and in thecourse of this replication the vector copies are distributedstatistically in the resulting cells depending on the number of thecopies present before and after cell division. Replication may takeplace in the nucleus of the host cell, and preferably replicates duringS-phase of the cell cycle. Moreover, the episomally replicating vectorcan be replicated at least once, i.e. one or multiple times, in thenucleus of the host cell during S-phase of the cell cycle.

The term “sample” is defined as any material to be tested in ananalytical or experimental method as described herein. Samples aretypically obtained from a subject as described herein. Samples include,but are not limited to, blood or blood fractions, saliva, urine, stool,cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breastmilk, synovial fluid, mucus (including rheum), tears, bile, gastricfluid, interstitial fluid, biopsies of tissues or epithelial cells thatare naturally shed or specifically collected from the body (such ascheek cell scrapings), aqueous humor, amniotic fluid, pleural fluid orbreath exhalation from a subject. In some embodiments, the sample isobtained via a non-invasive method (e.g. is a non-invasive sample).Exemplary non-invasive methods include but are not limited to passivecollection of bodily fluids, or non-injurious scrapings of tissuesaccessible to the external environment (e.g. of the epidermis, ormouth). Exemplary non-invasive samples include but are not limited tosaliva, sputum, mucus, sweat, urine, stool, semen, cervicovaginalsecretions, breast milk, rheum, tears, or cheek epithelial swabs. Insome embodiments, the sample is obtained via a minimally-invasivemethod. Exemplary minimally-invasive methods include, but are notlimited to, capillary collection, venipuncture, thoracentesis,amniocentesis, needle aspiration, or gastric lavage. Exemplaryminimally-invasive samples include, but are not limited to, blood orblood fractions (e.g. plasma or PBMC preparations), interstitial fluid,bile, gastric fluid, and amniotic fluid. In some embodiments, the sampleis obtained via biopsy. Exemplary biopsy samples include, but are notlimited to, skin biopsy samples (e.g. obtained by punch, shave,saucerization, wedge, incisional, or excisional biopsy), a bone marrowsamples (e.g. obtained by aspiration biopsy), a lymph node or breastbiopsies (e.g. obtained by fine-needle aspiration, core needle biopsy,vacuum assisted biopsy, or image-guided biopsy), a surgical biopsysamples (e.g. of an internal organ obtained by excisional or incisionalbiopsy), or mouth, GI-tract, lung, bladder, or urinary tract biopsysamples (e.g. obtained by endoscopy).

The term “origin of replication” as used herein refers to a DNA sequencethat is recognized by a replication initiation factor or a DNA replicaseleading to replication of a plasmid containing the origin ofreplication. The expression “recognized by a replication initiationfactor” is intended to mean that a replication initiation factor canphysically interact with all or a portion of an origin of replicationsequence, thereby causing or stimulating molecular mechanisms thatultimately cause all or a portion of the DNA molecule comprising theorigin of replication to be replicated. The origin of replication, thus,typically comprises functionally required elements. One example for suchfunctionally required elements are the family of repeats (FR) element orthe dyad symmetry (DS) element of the EBV origin of replication (OriP).Further origin of replications comprising functionally required elementsare well known in the art and are described for example in Bode et al.,(2001) Gene Ther. Mol. Biol. 6: 33-46. The parental nucleic acid plasmidvectors of the disclosure preferably comprise at least one origin ofreplication.

A “vector” is a nucleic acid sequence capable of transferring otheroperably-linked heterologous or recombinant nucleic acid sequences totarget cells. In some examples, a vector is a minicircle, plasmid,nanoplasmid, yeast artificial chromosome (YAC), bacterial artificialchromosome (BAC), cosmid, phagemid, bacteriophage genome, or baculovirusgenome. Suitable vectors also include vectors derived frombacteriophages or plant, invertebrate, or animal (including human)viruses such as CELiD vectors, adeno-associated viral vectors (e.g.AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotypedcombinations thereof such as AAV2/5, AAV2/2, AAV-DJ, or AAV-DJ8),retroviral vectors (e.g. MLV or self-inactivating or SIN versionsthereof, or pseudotyped versions thereof), herpesviral (e.g. HSV- orEBV-based), lentiviral vectors (e.g. HIV-, FIV-, or EIAV-based, orpseudotyped versions thereof), or adenoviral vectors (e.g. Ad5-based,including replication-deficient, replication-competent, orhelper-dependent versions thereof). In some embodiments, a vector is areplication competent viral-derived vector. In some embodiments, avector is a replication-incompetent viral-derived vector. In some cases,the vector may comprise an episomal maintenance element to facilitatereplication in one or more target cell type, such as a Scaffold/MatrixAttachment Region (S/MAR). S/MAR elements are particularly useful tofacilitate replication in the context of “naked” nucleic acid vectorssuch as minicircles. Exemplary suitable S/MAR elements include. but arenot limited to, EμMAR from the immunoglobulin heavy chain locus, theapoB MAR from the human apolipoprotein B locus, the Ch-LysMAR from thechicken lysozyme locus, and the huIFNβ MAR from the human IFNβ-locus. Avector may comprise a coding sequence capable of being expressed in atarget cell. Accordingly, as used herein, the terms “vector construct,”“expression vector,” and “gene transfer vector,” may refer to anynucleic acid construct capable of directing the expression of a gene ofinterest and which is useful in transferring the gene of interest intotarget cells. Vectors as described herein may additionally comprise oneor more cis-acting elements to stabilize or improve expression of mRNAstherefrom. Such cis-acting elements include but are not limited to anyof the elements described e.g., in Johansen et al. The Journal of GeneMedicine. (5)12:1080-1089 (doi: 10.1002/jgm.444) or Vlasova-St. Louisand Sagarsky. Mammalian Cis-Acting RNA Sequence Elements (doi:10.5772/intechopen.72124).

As one of the forms of vectors, the term “minicircle” as used hereinrefer to a small, double stranded circular DNA molecule that providesfor persistent, high level expression of a sequence of interest that ispresent on the vector, which sequence of interest may encode apolypeptide, an shRNA, an anti-sense RNA, an siRNA, and the like. Thesequence of interest is operably linked to regulatory sequences presenton the minicircle vector, said regulatory sequences controlling itsexpression. Such minicircle vectors are described, for example inpublished U.S. Patent Application US20040214329, herein specificallyincorporated by reference. As a different form of vectors, “nanoplasmid”refers to a vector that may comprise minimized bacterial ColE1 or R6Korigin of replication (which provides for such nanoplasmids to bereplicable in a bacterial host strain), a bacterial RNA-selectablemarker, and a eukaryotic gene region. Such nanoplasmids can comprise themini-R6K origin of SEQ ID NO: 3 and/or the RNA-OUT selectable marker ofSEQ ID NO: 4. Further examples of such elements (nanoplasmid origins andRNA-out selectable markers) are described e.g., in U.S. Pat. No.9,737,620B2, which is incorporated by reference herein for the purposesof describing nanoplasmid sequence elements.

The overall length of a minicircle vector is sufficient to include thedesired elements as described below, but not so long as to prevent orsubstantially inhibit to an unacceptable level the ability of the vectorto enter a target cell upon contact with the cell, e.g., via systemadministration to the host comprising the cell. As such, the minicirclevector can be generally at least about 0.3 kb long, often at least about1.0 kb long, whereas the parental vector may be as long as 6 kb, 10 kb,or longer.

Minicircle vectors differ from bacterial plasmid vectors in that theylack an origin of replication or lack a natural origin of replication(e.g. may comprise a minimized synthetic bacterial origin ofreplication), and lack a selection marker commonly found in bacterialplasmids, e.g. p-lactamase, tetracycline-resistance (tet),kanamycin-resistance (kan), or other antibiotic selection markers.Consequently, a minicircle becomes smaller in size, allowing moreefficient delivery. Minicircles lack the transgene expression silencingeffect which is associated with the vector backbone nucleic acidsequences of parental plasmids from which the minicircle vectors areexcised. The minicircle may be substantially free of vector sequencesother than the recombinase hybrid product sequence, and the sequence ofinterest, i.e. a transcribed sequence and regulatory sequences requiredfor expression.

The term “nanoplasmid” as used herein refer to a vector that maycomprise minimized bacterial ColE1 or R6K origin of replication (whichprovides for such nanoplasmids to be replicable in a bacterial hoststrain), a bacterial RNA-selectable marker, and a eukaryotic generegion. Some embodiments of nanoplasmids are described in e.g.US20150275221A1. In some embodiments, the nanoplasmid may comprise afusion bacterial-RNA-selectable marker/minimized origin of replication.In some embodiments, the fusion bacterial-RNA-selectablemarker/minimized origin of replication may be located within a syntheticintron located within the eukaryotic gene region of the nanoplasmid.

An RNA selectable marker is a vector-borne expressed non translated RNAthat regulates a chromosomally expressed target gene to afford selectionof the vector. This may be a plasmid borne nonsense suppressing tRNAthat regulates a nonsense suppressible selectable chromosomal target asdescribed by Crouzet J and Soubrier F 2005 U.S. Pat. No. 6,977,174included herein by reference. This may also be a plasmid borne antisenserepressor RNA, an RNA-OUTgene that represses RNA-IN regulated targets,pMB1 plasmid origin encoded RNAI that represses RNAII regulated targets,IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulatedtargets, ParB locus Sok of plasmid RI that represses Hok regulatedtargets, Flm locus FlmB of F plasmid that represses flmA regulatedtargets, another natural antisense repressor RNA such as those describedin e.g. Wagner E G H, Altuvia S, Romby P. 2002. Adv Genet 46:361 andFranch T, and Gerdes K. 2000. Current Opin Microbiol 3: 159, or anengineered repressor RNA such as a small synthetic small RNA like theSgrS, MicC or MicF scaffolds as described in Park et al. NatureBiotechnology volume 31, pages 170-174 (2013).

A number of suitable methods for transfecting cells according to thedisclosure are available. By “transfected” it is meant an alteration ina cell resulting from the uptake of foreign nucleic acid, usually DNA.Use of the term “transfection” is not intended to limit introduction ofthe foreign nucleic acid to any particular method. Thus, suitablemethods include viral infection/transduction, conjugation, nanoparticledelivery, electroporation, particle gun technology, calcium phosphateprecipitation, direct microinjection, and the like. The choice of methodis dependent on the type of cell being transfected and the circumstancesunder which the transfection is taking place (i.e. in vitro, ex vivo, orin vivo). A general discussion of these methods can be found in Ausubel,et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons,1995, which are hereby incorporated by reference.

The term “transfection agent” may encompass any compound that mediatesincorporation of DNA or RNA into a host cell, e.g., a liposome. Suitablemethods for transforming or transfecting host cells can be found inSambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), Ausubel, et al., Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995, and other laboratorymanuals, which are hereby incorporated by reference. Examples ofsuitable transfection agents include, but are not limited to, linear orbranched polyethylenimines, nanoparticles, liposomes, lipophilicparticles, solid nanoparticles, amphipathic peptides, micelles,dendrimers, polymeric compositions, hydrogels, synthetic or naturallyderived exosomes, virus-like particles, or any combination thereof.

The term “EXOmotif”, as used herein, refers to an RNA sequencecontrolling loading of a miRNA into an exosome. In some embodiments, anEXOmotif may mediate the binding of a miRNA to heterogeneousribonucleoprotein A2B1 (hnRNPA2B1), which has been described ascontrolling the loading of miRNAs into exosomes. Such sequences include,but are not limited to, 5′-GGAG-3′ and 5′-CCCU-3′.

The terms “nucleic acid molecule” and “polynucleotide” as used hereinrefer polymeric forms of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,shRNA, single-stranded short or long RNAs, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, control regions, isolated RNA of any sequence, nucleic acidprobes, and primers. The nucleic acid molecule may be linear orcircular.

The term “promoter” is a DNA sequence that directs the transcription ofa polynucleotide. Typically, a promoter can be located in the 5′ regionof a polynucleotide to be transcribed, proximal to the transcriptionalstart site of such polynucleotide. More typically, promoters are definedas the region upstream of the first exon; more typically, as a regionupstream of the first of multiple transcription start sites. Frequentlypromoters are capable of directing transcription of genes located oneach of the complementary DNA strands that are 3′ to the promoter.Stated differently, many promoters exhibit bidirectionality and candirect transcription of a downstream gene when present in eitherorientation (i.e. 5′ to 3′ or 3′ to 5′ relative to the coding region ofthe gene). Additionally, the promoter may also include at least onecontrol element such as an upstream element. Such elements includeupstream activator regions (UARs) and optionally, other DNA sequencesthat affect transcription of a polynucleotide such as a syntheticupstream element.

The terms “coding sequence” and “encodes” when used in reference to apolypeptide herein refer to a nucleic acid molecule that is transcribed(in the case of DNA) and translated (in the case of mRNA) into apolypeptide, for example, when the nucleic acid is present in a livingcell (in vivo) and placed under the control of appropriate regulatorysequences (or “control elements”). The boundaries of the coding sequenceare typically determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from viral, prokaryotic oreukaryotic mRNA, genomic DNA sequences from viral, eukaryotic, orprokaryotic DNA, and synthetic DNA sequences. A transcriptiontermination sequence may be located 3′ to the coding sequence, and apromoter may be located 5′ to the coding sequence; along with additionalcontrol sequences if desired, such as enhancers, introns, polyadenylation site, etc. A DNA sequence encoding a polypeptide may beoptimized for expression in a selected cell by using the codonspreferred by the selected cell to represent the DNA copy of the desiredpolypeptide coding sequence.

The term “barcode” or “barcode molecule” as used herein generally refersto a label, or an identifier, that conveys or is capable of conveyinginformation about a molecule to which the barcode/barcode molecule isattached. A barcode/barcode molecule may be unique. Barcodes/barcodemolecules may have a variety of different formats. For example,barcodes/barcode molecules can include polynucleotide barcodes; randomnucleic acid and/or amino acid sequences; and synthetic nucleic acidand/or amino acid sequences. A barcode/barcode molecule can be attachedto a molecule in a reversible or irreversible manner. A barcode can beadded to, for example, a fragment of a deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sample before, during, and/or after sequencing ofthe sample. Barcodes can allow for identification and/or quantificationof individual sequencing-reads.

The term “operably linked” as used herein refers to an arrangement ofelements wherein the components so described are configured so as toperform their usual function. Thus, a given promoter that is operablylinked to a coding sequence (e.g., a reporter expression cassette) iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter or other control elements neednot be contiguous with the coding sequence, so long as they function todirect the expression thereof. For example, intervening untranslated yettranscribed sequences can be present between the promoter sequence andthe coding sequence and the promoter sequence can still be considered“operably linked” to the coding sequence.

The term “expression cassette” as used herein refers to any nucleic acidconstruct capable of directing the expression of any RNA transcriptincluding gene/coding sequence of interest as well as non-translatedRNAs, such as shRNAs, microRNAs, siRNAs, anti-sense RNAs, and the like.Such cassettes can be constructed into a “vector,” “vector construct,”“expression vector,” or “gene transfer vector,” in order to transfer theexpression cassette into target cells. Thus, the term includes cloningand expression vehicles, as well as viral vectors.

The term “target cell” as used herein refers to a cell that in which agenetic modification is desired. Target cells can be isolated (e.g., inculture) or in a multicellular organism (e.g., in a blastocyst, in afetus, in a postnatal animal, and the like).

The term “pharmaceutically acceptable carrier” as used herein refers toa diluent, adjuvant, excipient, or vehicle with which a probe of thedisclosure is administered and which is approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as pea-nut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. When administered to a patient, the probe andpharmaceutically acceptable carriers can be sterile. Water is a usefulcarrier when the probe is administered intravenously. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Suitablepharmaceutical carriers also include excipients such as glucose,lactose, sucrose, glycerol monostearate, sodium chloride, glycerol,propylene, glycol, water, ethanol and the like. The presentcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. The present compositionsadvantageously may take the form of solutions, emulsion,sustained-release formulations, or any other form suitable for use.

The term “detectable” refers to the ability to detect a signal over thebackground signal. The detectable signal is defined as an amountsufficient to yield an acceptable image using equipment that isavailable for pre-clinical use. A detectable signal maybe generated byone or more administrations of the probes of the present disclosure. Theamount administered can vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, the dosimetry,and the like. The amount administered can also vary according toinstrument and digital processing related factors.

The term “in vivo imaging” as used herein refers to methods or processesin which the structural, functional, or physiological state of a livingbeing is examinable without the need for a life-ending sacrifice.

The term “non-invasive in vivo imaging” as used herein refers to methodsor processes in which the structural, functional, or physiological stateof a being is examinable by remote physical probing without the need forbreaching the physical integrity of the outer (skin) or inner(accessible orifices) surfaces of the body.

The “imaging moiety” may be detected either externally to a subjecthuman or non-human animal body or via use of detectors designed for usein vivo, such as intravascular radiation or optical detectors such asendoscopes, or radiation detectors designed for intra-operative use. Theimaging moiety is preferably but is not limited to a reporter suitablefor in vivo optical imaging.

The term “bioluminescence” as used herein refers to a type ofchemiluminescent emission of light by biological molecules, particularlyproteins. The essential condition for bioluminescence is molecularoxygen, either bound or free in the presence of an oxygenase, aluciferase, which acts on a substrate, a luciferin in the presence ofmolecular oxygen and transforms the substrate to an excited state, whichupon return to a lower energy level releases the energy in the form oflight.

The term “luciferase” as used herein refers to oxygenases that catalyzea light emitting reaction. For instance, bacterial luciferases catalyzethe oxidation of flavin mono-nucleotide and aliphatic aldehydes, whichreaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina luciferin, andanother class of luciferases catalyzes the oxidation of Coleopteraluciferin. Thus, “luciferase” refers to an enzyme or photoprotein thatcatalyzes a bioluminescent reaction. The luciferases such as firefly andRenilla luciferases are enzymes that act catalytically and are unchangedduring the bioluminescence generating reaction. The luciferasephotoproteins, such as the aequorin and obelin photoproteins to whichluciferin is non-covalently bound, are changed by release of theluciferin, during bioluminescence generating reaction. The luciferase isa protein that occurs naturally in an organism or a variant or mutantthereof, such as a variant produced by mutagenesis that has one or moreproperties, such as thermal or pH stability, that differ from thenaturally-occurring protein. Luciferases and modified mutant or variantforms thereof are well known. Reference, for example, to “Renillaluciferase” means an enzyme isolated from member of the genus Renilla oran equivalent molecule obtained from any other source, such as fromanother Anthozoa, or that has been prepared synthetically.

“Bioluminescent protein” refers to a protein capable of acting on abioluminescent initiator molecule substrate to generate or emitbioluminescence.

“Bioluminescent initiator molecule” is a molecule that can react with abioluminescent donor protein to generate bioluminescence. Thebioluminescence initiator molecule includes, but is not limited to,coelenterazine, analogs thereof, and functional derivatives thereof.Derivatives of coelenterazine include, but are not limited to,coelenterazine 400a, coelenterazine cp, coelenterazine f, coelenterazinefcp, coelenterazine h, coelenterazine hcp; coelenterazine ip,coelenterazine n, coelenterazine 0, coelenterazine c, coelenterazine c,coelenterazine coelenterazine icp, coelenterazine 2-methyl,benzyl-coelenterazine bisdeoxycoelenterazine, and deep bluecoelenterazine (DBC) (described in more detail in U.S. Pat. Nos.6,020,192; 5,968,750 and 5,874,304).

In general, coelenterazines are known to luminesce when acted upon by awide variety of bioluminescent proteins, specifically luciferases.Useful, but non-limiting, coelenterazines are disclosed in U.S. patentapplication Ser. No. 10/053,482, filed Nov. 2, 2001, the disclosure ofwhich is hereby incorporated by reference in its entirety.Coelentera-zines are available from Promega Corporation, Madison, Wis.and from Molecular Probes, Inc., Eugene, Oreg. Coelentera-zines may alsobe synthesized as described for example in Shimomura et al., (1989)Biochem. J. 261: 913-920; Inouye et al., (1997) Biochem. Biophys. Res.Comm. 233: 349-353, 1997; and Teranishi et al., (1997) Anal. Biochem.249: 37-43.

The term “Survivin” as used herein refers to a protein also calledbaculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, is aprotein that, in humans, is encoded by the BIRC5 gene. (NCBI ReferenceSequence: NG 029069.1). Survivin is a member of the inhibitor ofapoptosis (IAP) family. The survivin protein inhibits caspaseactivation, thereby leading to negative regulation of apoptosis orprogrammed cell death. This has been shown by disruption of survivininduction pathways leading to an increase in apoptosis and decrease intumor growth. The survivin protein is expressed highly in most humantumors and fetal tissue but is completely absent in terminallydifferentiated cells. Survivin expression is also highly regulated bythe cell cycle and is only expressed in the G2-M phase. It is known thatsurvivin localizes to the mitotic spindle by interaction with tubulinduring mitosis and may play a contributing role in regulating mitosis.Regulation of survivin seems to be linked to the p53 protein. It also isa direct target gene of the Wnt pathway and is upregulated by β-catenin.

It is contemplated, however, that the minicircles of the disclosure mayutilize any tumor-specific promoter operably linked to a reporter orother heterologous nucleic acid sequence desired to be expressed in atarget cell. For example, but not intended to be limiting, suitablepromoters known in the art include: CXCR4 promoter tumor-specific inmelanomas; Hexokinase type II promoter tumor-specific in lung cancer;TRPM4 (Transient Receptor Potential-Melastatin 4) promoter ispreferentially active in prostate cancer; stromelysin 3 promoter isspecific for breast cancer cells (Basset et al., (1990) Nature 348:699); surfactant protein A promoter specific for non-small cell lungcancer cells (Smith et al., 1994) Hum. Gene Ther. 5: 29-35); secretoryleukoprotease inhibitor (SLPI) promoter specific for SLPI-expressingcarcinomas (Garver et al., (1994) Gene Ther. 1: 46-50); tyrosinasepromoter specific for melanoma cells (Vile et al., (1994) Gene Ther. 1:307); stress-inducible grp78/BiP promoter specific forfibrosarcoma/tumorigenic cells (Gazit et al., (1995) Cancer Res. 55:1660); interleukin-10 promoter specific for glioblastoma multiform cells(Nitta et al., (1994) Brain Res. 649: 122); a-B-crystallin/heat shockprotein 27 promoter specific for brain tumor cells (Aoyama et al.,(1993) Int. J. Cancer 55: 760); epidermal growth factor receptorpromoter specific for squamous cell carcinoma, glioma, and breast tumorcells (Ishii et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 282);mucin-like glycoprotein (DF3, MUC1) promoter specific for breastcarcinoma cells (Abe et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90:282); mts 1 promoter specific for metastatic tumors (Tulchinsky et al.,(1992) Proc. Natl. Acad. Sci. U.S.A. 89: 9146); NSE promoter specificfor small-cell lung cancer cells (Forss-Petter et al., (1990) Neuron 5:187); somatostatin receptor promoter specific for small cell lung cancercells (Bombardieri et al., (1995) Eur. J. Cancer 31A: 184; Koh et al.,(1995) Int. J. Cancer 60: 843); c-erbB-3 and c-erbB-2 promoters arespecific for breast cancer cells (Quin et al., (1994) Histopathology 25:247); c-erbB4 promoter specific for breast and gastric cancer cells(Rajkumar et al., (1994) Breast Cancer Res. Trends 29: 3); thyroglobulinpromoter specific for thyroid carcinoma cells (Mariotti et al., (1995)J. Clin. Endocrinol. Meth. 80: 468); a-fetoprotein promoter specific forhepatoma cells (Zuibel et al., (1995) J. Cell. Phys. 162: 36); villinpromoter specific for gastric cancer cells (Osborn et al., (1988)Virchows Arch. A. Pathol. Anat. Histopathol. 413: 303); and albuminpromoter specific for hepatoma cells (Huber, (1991) Proc. Natl. Acad.Sci. U.S.A. 88: 8099), which are all hereby incorporation by reference.Other examples of promoters are an ATP binding cassette subfamily Cmember 4 (ABCC4) promoter, an anterior gradient 2, protein disulphideisomerase family member (AGR2) promoter, activation induced cytidinedeaminase (AICDA) promoter, an UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin3 (CDH3) promoter, a CEA cell adhesion molecule 5 (CEACAM5) promoter, acentromere protein F (CENPF) promoter, a centrosomal protein 55 (CEP55)promoter, a claudin 3 (CLDN3) promoter, a claudin 4 (CLDN4) promoter, acollagen type XI alpha 1 chain (COL11A1) promoter, a collagen type Ialpha 1 chain (COL1A1) promoter, a cystatin SN (CST1) promoter, adenticleless E3 ubiquitin protein ligase homolog (DTL) promoter, afamily with sequence similarity 111 member B (FAM111B) promoter, aforkhead box A1 (FOXA1) promoter, a kinesin family member 20A (KIF20A),a laminin subunit gamma 2 (LAMC2) promoter, a mitotic spindlepositioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group IID (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a ubiquitin conjugating enzyme E2 T(UBE2T) promoter, a checkpoint kinase 1 (CHEK1) promoter, an epithelialcell transforming 2 promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter,a centromere protein I (CENPI) promoter, an E2F transcription factor 1(E2F1) promoter, a flavin adenine dinucleotide synthetase 1 (FLAD1)promoter, a protein phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G)promoter, an ubiquitin conjugating enzyme E2 S (UBE2S) promoter, anaurora kinase A and ninein interacting protein (AUNIP) promoter, a celldivision cycle 6 (CDC6) promoter, a centromere protein L (CENPL)promoter, a DNA replication helicase/nuclease 2 (DNA2) promoter, a DSN1homolog, MIS12 kinetochore complex component (DSN1) promoter, adeoxythymidylate kinase (DTYMK) promoter, a G protein regulated inducerof neurite outgrowth 1 (GPRIN1) promoter, a mitochondrial fissionregulator 2 (MTFR2) promoter, a RAD51 associated protein 1 (RAD51AP1)promoter, a small nuclear ribonucleoprotein polypeptide A′ (SNRPA1)promoter, an ATPase family, AAA domain containing 2 (ATAD2) promoter, aBUB1 mitotic checkpoint serine/threonine kinase (BUB1) promoter, acalcyclin binding protein (CACYBP) promoter, a cell division cycleassociated 3 (CDCA3) promoter, a centromere protein O (CENPO) promoter,a flap structure-specific endonuclease 1 (FEN1) promoter, a forkhead boxM1 (FOXM1) promoter, a cell proliferation regulating inhibitor ofprotein phosphatase 2A (KIAA1524) promoter, a kinesin family member 2C(KIF2C) promoter, a karyopherin subunit alpha 2 (KPNA2) promoter, a MYBproto-oncogene like 2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2)promoter, a RAN binding protein 1 (RANBP1) promoter, a small nuclearribonucleoprotein polypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80kinetochore complex component (SPC24) promoter, a transforming acidiccoiled-coil containing protein 3 (TACC3) promoter, a TBC1 domain familymember 31 (TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, azinc finger protein 695 (ZNF695) promoter, an aurora kinase A (AURKA)promoter, a BLM RecQ like helicase (BLM) promoter, a chromosome 17 openreading frame 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, acyclin B1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F(CCNF), a cell division cycle 20 (CDC20) promoter, a cell division cycle45 (CDC45) promoter, a cell division cycle associated 5 (CDCA5)promoter, a cyclin dependent kinase inhibitor 3 (CDKN3) promoter, acadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) promoter, acentromere protein A (CENPA) promoter, a centrosomal protein 72 (CEP72)promoter, a CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, acollagen type X alpha 1 chain (COL10A1) promoter, a chromosomesegregation 1 like (CSE1L) promoter, a DBF4 zinc finger promoter, a GINScomplex subunit 1 (GINS1) promoter, a G protein-coupled receptor 19(GPR19) promoter, a kinesin family member 18A (KIF18A) promoter, akinesin family member 4A (KIF4A) promoter, a kinesin family member C1(KIFC1) promoter, a minichromosome maintenance 10 replication initiationfactor (MCM10) promoter, a minichromosome maintenance complex component2 (MCM2) promoter, a minichromosome maintenance complex component 7(MCM7) promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, a functional fragment thereof, or any combination thereof.

Further definitions are provided in context below. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art ofmolecular biology. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described herein.

Abbreviations

SEAP, Secreted Embryonic Alkaline Phosphatase; MRI, magnetic resonanceimaging; SPECT, Single-photon emission computed tomography; MC,mini-circle; PP, parental plasmid; WPRE, Woodchuck Hepatitis Virus (WHP)Post-transcriptional Regulatory Element (WPRE; Luc, luciferase; BLI,bioluminescence imaging; ROI, region of interest; AUC, area under thecurve; RG, reporter gene; TS, tumor-specific; Fluc (FLUC), fireflyluciferase, ROC (receiver operator-characteristic)

INTRODUCTION

Early detection of cancer can dramatically improve the efficacy ofavailable treatment strategies. Yet, despite decades of effort onblood-based biomarker cancer detection, many promising endogenousbiomarkers have failed clinically due to intractable problems such ashighly variable background expression from non-malignant tissues.Strategies for improved cancer diagnosis have traditionally relied onmeasurement of endogenous molecules that are over-expressed in cancercells either via molecular imaging or blood-based assays. A challenge ofthese strategies is often significant expression within non-canceroustissues, leading to high background levels and confounding results. Analternative strategy is to utilize promoters of tumor-specific (TS)proteins in exogenously-delivered gene vectors in order to drive theexpression of unique reporter genes (RGs) strictly within tumors. Forthis strategy to become a reality, safety, specificity, and sensitivityare of utmost importance. While safer than viral vectors, two drawbacksof non-viral vectors have been low gene transfer rates and transientexpression profiles. Minicircles (MCs) are plasmids that lack abacterial backbone and are advantageous to overcome the above keyissues.

The present disclosure provides embodiments of an alternative andadvantageous detection strategy based on systemic administration ofsafe, tumor-activatable minicircles that utilize the pan-tumor-specificSurvivin promoter to drive expression of a secretable reporter gene thatis detectable in the blood near-exclusively in tumor-bearing subjects.After systemic administration a robust ability to differentiate micebearing experimental human melanoma metastases from tumor-free subjectsfor up to 2 weeks simply by measuring blood reporter levels has beenshown. Cumulative changes in reporter levels also identifiedtumor-bearing subjects, and a receiver operator-characteristic curveanalysis highlighted this test's performance with an AUC of 0.918±0.084.Lung tumor burden correlated (r2=0.714; p<0.05) with cumulative reporterlevels indicating that a determination of disease extent was possible.Continued development of our system could dramatically improve tumordetectability due to temporally-controlled, high reporter expression intumors and near-zero background from healthy tissues is possible.

Tumor-specific nanoplasmid vectors driving the expression of eithersecreted embryonic alkaline phosphatase (SEAP) or firefly luciferase(FLUC) have been developed and their utility validated for detectingtumors after systemic administration using blood- and/or imaging-basedassays. For gene vectors to be used for cancer screening purposes,challenges include efficient tumor delivery, achieving potent expressionfor maximum sensitivity, stringent control of expression to attain tumorspecificity, and minimization of safety concerns. Tumor-specificminicircle vectors can overcome all of these challenges and it is nowshown that systemically administered tumor-specific minicircle vectorscan be assayed via serum and non-invasive imaging to differentiallyidentify tumor-bearing subjects from normal subjects. Importantly, thetumor-specific minicircle vectors of the disclosure advantageously havebroad applicability in many patient populations since the Survivinpromoter drives expression across many different tumor types of tumorcell. The tumor-specific minicircle vectors of the disclosure provide anovel cancer management paradigm that involves tumor detection via aninitial blood-based assay, tumor localization via molecular-geneticimaging, and tumor treatment using theranostic tumor-specific minicirclevectors.

The present disclosure encompasses embodiments of nucleic acidnanoplasmid vectors most advantageous for the detection of tumor cells.In particular, the minicircles of the disclosure incorporate atumor-specific promoter operably linked to a nucleotide sequence desiredto be selectively expressed in a tumor cell or a tissue comprising apopulation of tumor cells. In some embodiments of the disclosure, theminicircle vectors comprise a tumor-specific promoter operably linked toa nucleotide sequence encoding a polypeptide useful as a reporter.Accordingly, when expressed by a recipient tumor cell, the reporter maybe detectable, thereby providing information such as a visual image ofthe tumor cell and/or its location in a tissue of the subject human ornon-human animal.

In some embodiments, the nanoplasmid vectors according to the disclosurecan advantageously deliver an expressible reporter gene to a tumor cell.It is within the scope of the disclosure for the reporter gene to bedetectable by such non-invasive detection methods as MRI imaging, PETimaging, SPECT imaging, luminescence imaging and the like. For example,but not intended to be limiting, MRI reporter genes encode for creatinekinase; tyrosinase; transferrin receptor; ferritin; Mag A. PET imagingreporter genes include, but are not limited to such as Herpes simplexvirus 1 thymidine kinase (HSV1-TK); hypoxanthine phosphoribosyltransferase; L-amino acid decarboxylase; dopamine 2 receptor (D2R,including the mutant D2RA80); somatostatin receptor; estrogen receptor(hERL); dopamine transporter; sodium iodide symporter; catecholaminetransporter; 13-galactosidase. PET/SPECT imaging reporter genes include,but are not limited to, Herpes simplex virus Type 1 thymidine kinase andmultiple optimized mutants, such as HSV1-sr39tk; dopamine type 2receptor; sodium iodide symporter; somatostatin type 2 receptor; humannorepinephrine transporter; human estrogen receptor a; mutants of humandeoxycytidine kinase; and recombinant carcinoembryonic antigen.Bioluminescence reporter genes include, but are not limited to, fireflyluciferase (f1); synthetic Renilla luciferase (hr1); Enhanced GreenFluorescence protein (egfp); Red Fluorescence Protein (rfp); monomericRed Fluorescence Protein (mrfp 1), and the like. It is further possiblefor the reporter genes suitable for incorporation into the minicirclesof the disclosure to provide multi-modality methods of imaging. Forexample, but not intended to be limiting, a reporter gene suitable forphotoacoustic, MRI, and PET imaging, is the gene encoding humantyrosinase, as described by Qin et al., (2013), Sci. Rpts. 3: Art. No.:1490, incorporated herein by reference its entirety.

In addition to the advantageous use of the nanoplasmids of thedisclosure for selectively detecting a recipient tumor cell, thenucleotide sequence operably linked to the tumor-specific promoter mayencode a polypeptide useful for modulating the proliferation ormetabolic activity of a recipient tumor cell for the purpose of reducingor eliminating the targeted tumor cell from the subject human ornon-human animal.

For example, but not intended to be limiting, therapeutically effectivepolypeptides that are advantageous for targeting and therapeuticallychallenging a tumor cell include HSVtk; cytosine deaminase; DTdiaphorase; nitroreductase; guanine phosphoribosyl transferase; purinenucleoside phosphorylase; thymidine phorphorylase; carboxylesterase;folylpolyglutamyl synthetase; carboxypeptidase A1; carboxypeptidase G2;cytochrome P-450 (CYP2B1), and the like. The activities of thesepolypeptides for the conversion of a prodrug to an effective therapeuticcomposition are described in, for example, Harrington et al., (2002)Clinical Oncology 14: 148-169 incorporated herein by reference in itsentirety.

In further embodiments of the disclosure, it is contemplated that thenucleotide sequence tumor-specifically expressed from the minicircle maynot be translated into a heterologous polypeptide but rather may beexpressed as a short interfering ribonucleotide sequence (siRNA) thatmay interact with at least one gene regulatory element of the recipienttumor cell, again modulating the proliferation or metabolic activity ofa recipient tumor cell. Alternatively, it is contemplated that thenucleotide sequence may be expressed as a microRNA sequence (miRNA) oras a synthetic RNA sequence that does not correspond to any knownendogenous sequence and only serves the purpose of being an agentdetectable by nucleic acid hybridization or amplification-basedtechniques (a nucleic acid biomarker).

Accordingly, it is contemplated to be within the scope of the disclosureto provide embodiments of nucleic acid minicircle vectors (and theparental plasmids thereof) useful for selectively targeting tumor cellscultured in vitro or, most advantageously, in vivo to obtain detectablesignals identifying and/or locating a cancerous cell or population oftumor cells in the subject as well as for delivering a therapeutic agent(peptide, polypeptide, nucleic acid) to the targeted tumor cells.

The present disclosure provides nucleic acid minicircle vectors usefulfor administering to a subject human or non-human animal for the purposeof detecting the presence of a targeted tumor cell or cells (including atumor tissue). For example, the minicircle constructMC-pSurv-SEAP-WPRE-SV40PolyA as shown in FIG. 3 and having thenucleotide sequence SEQ ID NO: 1 as shown in FIG. 13, comprises anucleic acid fragment encoding the detectable polypeptide secretedembryonic alkaline phosphatase (SEAP) operably linked to thetumor-specific promoter pSurvivin.

When delivered to cultured melanoma cells, to subcutaneous melanomaxenografts, or intravenously to animals that have a developed tumor, theminicircle vectors of the disclosure provide detectable signals, eitheras a serum secreted alkaline phosphatase polypeptide or as abioluminescent signal in the minicircle vector construct where SEAP hadbeen replaced by a luciferase reporter, as shown in FIG. 4 (and havingthe nucleotide sequence SEQ ID NO: 2 as shown in FIG. 14). Accordingly,it has been demonstrated that the minicircle constructs of thedisclosure can identify, in the recipient animal or human, bothmetastatic tumor cells or a localized tumor.

The present disclosure further provides methods of modulating thephysiology or proliferation of a targeted tumor cell by delivering aminicircle nucleic acid to said tumor cell, allowing the targeted cellto express the nucleotide sequence from the nucleic acid sequenceoperably linked to the tumor-specific promoter, and allowing theexpressed product to interact with the targeted cell, thereby modifyingthe physiological status of the cell or cells.

In a first instance, the disclosure provides embodiments of nucleic acidminicircles wherein a tumor-specific promoter is such as, but notlimited to, the Survivin promoter.

Accordingly, to overcome the limitations of endogenous biomarkerdetection, the disclosure provides embodiments of a strategy based onidentification of tumor-bearing individuals using blood-based detectionof exogenously delivered genetically-encoded reporters, which producetumor-driven biomarkers. The main advantage of this strategy is theability to tailor biomarker expression exclusively in cells of aparticular phenotype (i.e. tumor cells), thereby reducing the number offalse positives due to protein production from non-malignant tissues.Thus, systemic administration of a tumor-activatable vector encoding asecretable reporter gene can be utilized to identify tumor-bearingsubjects provided that transgene expression was transcriptionallytargeted to cancer cells using a tumor-specific promoter (a promoter ofa gene expressing a protein that is only present in tumors), as shown inFIG. 1. For this strategy to be translated into the clinic, the safety,specificity, sensitivity, and broad applicability are important and eachcomponent of the systems of the disclosure were chosen to offer maximumtranslational potential. Specifically, the present disclosure providesnon-viral tumor-activatable minicircles (MCs) encoding a reporter geneincluding, but not limited to, human secreted embryonic alkalinephosphatase (SEAP) that attain tumor specificity through the use of atumor-specific promoter such as, but not limited to, the Survivinpromoter (pSurv).

While safer than viral vectors, two drawbacks of traditional non-viralvectors (i.e. plasmids) are low gene transfer rates and transientexpression profiles. MCs are essentially plasmids that lack theprokaryotic backbone required only for expansion in bacteria. MCs haverepeatedly shown to demonstrate improved expression profiles (months innon-dividing, and weeks in dividing, cells) compared to their plasmidcounterparts due to their smaller size and reduced promoter silencing(Darquet et al., (1997) Gene Therapy 4: 1341-1349; Darquet et al.,(1999) Gene Therapy 6: 209-218; Chen et al., (2003) Mol. Therapy: J. Am.Soc. Gene Therapy 8: 495-500; Chen et al., (2004) Gene Therapy 11:856-864). MCs also conform to regulatory “plasmids free of antibioticresistance genes” (pFAR) principles (Marie et al., (2010) J. Gene Med.12: 323-332) which are known to be safer for human administration thanconstructs containing antibiotic resistance genes. Moreover, whileproducing MCs was traditionally very labor-intensive and time-consuming,more recent advances in MC production schemes have made it possible toproduce large quantities in short periods of time with relative ease andreduced costs (Kay et al., (2010) Nat. Biotech. 28: 1287-1289). Finally,while integration is a safety concern with many gene (particularlyviral) vectors, even with effective in vivo delivery methods like directlocal injection and electroporation, the integration rates of non-viralvectors are approximately 1-3 orders of magnitude below the rate ofspontaneous gene-inactivating mutations (Wang et al., (2004) GeneTherapy 11: 711-721; Nichols et al., (1995) Annals New York Acad. Sci.772: 30-39; Ledwith et al., (2000) Develop. Biologicals 104: 33-43;Ledwith et al., (2000) Intervirology 43: 258-272). Hence, MCs havebecome one of the most useful non-viral vector platforms in terms oftranslational potential, potency and safety.

SEAP is a commonly used secretable reporter protein and has many idealcharacteristics. It is an artificial, C-terminal truncated, secretableform of human placental alkaline phosphatase (PLAP) that is onlyexpressed during embryogenesis; thus, it is a unique reporter notnormally found in the blood and should have near-zero background (Bergeret al., (1988) Gene 66: 1-10). Compared to PLAP, SEAP is unusuallyheat-stable; thus, heating samples to 65° C. allows SEAP to bespecifically assayed (Bronstein et al., (1994) BioTechniques 17:172-174, 76-177). Commercial SEAP detection assays are extremelysensitive over at least a 4-log order concentration range, withdetection limits in the picogram/ml range. SEAP is also an advantageousprotein-based reporter for translation into the clinic since: 1) it hasshown effective longitudinal monitoring of non-viral gene transfer inmice and large animals (Brown et al., (2008) Methods Mol. Biol. 423:215-224); 2) its human origin implies it can have reduced or zeroimmunogenic potential in patients similar to what has been shown withmurine SEAP (mu-SEAP) in immunocompetent mice (Wang et al., (2001) Gene279: 99-108); and 3) SEAP has been used in the clinic to monitorantibody levels following administration of an HPV16/18 AS04-adjuvantedvaccine (Kemp et al., (2008) Vaccine 26: 3608-3616).

The systems of the disclosure utilize pSury to drive the expression ofSEAP. Survivin is a member of the apoptosis inhibitor family that helpscontrol mitotic progression and prevent cell death and is over-expressedin many cancers such as melanoma, liver, lung, breast, colon andovarian, but not in healthy adult tissues (Ito et al., (2000) Hepatology31: 1080¬1085; Chen et al., (2004) Cancer Gene Therapy 11: 740-747; Luet al., (2005) Gene Therapy 12: 330-338). pSurv, therefore, isadvantageous for transcriptional targeting of tumors as demonstrated inmodels of lung, melanoma, colon, breast, ovarian, and liver cancer (Luet al., (2005) Gene Therapy 12: 330-338; Li et al., (2006) J. Gene Med.8: 1232-1242; van Houdt et al., (2006) J. Neurosurgery 104: 583-592; Ahnet al., (2011) Gene Therapy 18: 606-612; Ray et al., (2008) Mol.Therapy: J. Am. Soc. Gene Therapy 16: 1848-1856). Thus, thetumor-specific promoter-driven tumor-activatable MCs of the disclosureoffer broad applicability for effective cancer screening across numeroustumor types and patient populations.

Accordingly, diagnostic tumor-activatable MCs have been developed andtested for the ability to distinguish tumor-bearing subjects fromhealthy subjects after systemic administration of the MCs by measuringblood levels of a genetically-encoded cancer biomarker. For delivery,the MCs were compared with a non-targeted transfection agent that hasbeen shown to have no immunogenicity (Bonnet et al., (2008) Pharmaceut.Res. 25: 2972-2982), the ability to repeatedly dose animals, and theability efficiently transfect both primary and metastatic tumors in miceafter systemic (tail-vein) administration (Yang et al., (2013) Proc.Nat. Acad. Sci. U.S.A. 110: 14717-14722; Bhang et al., (2011) Nat. Med.17: 123-129). The results indicate that use of tumor-activatable MCs isan advantageous promising platform technology for safe and efficaciouscancer screening. This system is useful for monitoring patients athigh-risk for tumor recurrence, followed by screening high-riskpopulations prior to tumor diagnosis, and can be advantageous forscreening for the general population.

An exogenously delivered genetically-encoded cancer blood biomarkervector strategy according to the disclosure can overcome some of theinherent limitations of cancer screening targeting endogenous cancerblood biomarkers such as high background expression in healthy tissuesand random fluctuations in biomarker expression over time. The presentdisclosure provides embodiments of a tumor-activatable MC system thatcan be administered systemically to identify tumor-bearing subjectsusing a simple and relatively inexpensive blood-based assay. The assayshowed reliable detection capabilities and assessment of disease extent,indicating the feasibility of tumor-activatable MCs as a highly robustand safe cancer screening system.

Research in cancer gene therapy has sought methods for expressing atherapeutic transgenes specifically within tumors to avoid undesirableeffects in non-target or normal cells. To reach this goal severalstrategies have been explored including transcriptional targeting oftumors using tumor-specific promoters (Aim et al., (2011) Gene Therapy18: 606-612; Ye et al., (2003) Biochem. Biophys. Res. Comms. 307:759-764; Iyer et al., (2005) Transgenic Res. 14:47-55), transcriptionalsilencing or repression in healthy tissues using endogenous miRNAregulation (Cawood et al., (2009) PLoS Pathogens 5: e 1 000440; Ronaldet al., (2013) Gene Therapy 20: 1006-1013), enhanced tumor tropism ofboth viral (transduction targeting) and non-viral vectors (Chisholm etal., (2009) Cancer Res. 69: 2655-2662; Bachtarzi et al., (2008) ExpertOpinion Drug Delivery 5: 1231-1240), or combinations of these strategies(Tsuruta et al., (2008) Clin. Cancer Res. 14: 3582-3588; Sugio et al.,(2011) Clin. Cancer Res 17: 2807-2818). The systems of the disclosureprovide a means of expressing a secretable reporter gene for thepurposes of cancer detection. With this application of gene vectorscomes the additional challenge of overcoming heightened safety concerns,since as a potential screening tool the vectors could be used inpatients without any clearly visible evidence of cancer. Therefore, allcomponents of this type of system need to be safe including the deliveryvehicle (if needed), the DNA vector itself, and the transgene (ifexpressed).

While many delivery formulations are known in the art and contemplatedfor use with the MC systems of the disclosure, an in vivo transfectionagent that has a desirable safety profile (i.e. no immunostimulation)(Bonnet et al., (2008) Pharmaceut. Res. 25: 2972-2982) and is in phaseI/II clinical trials (Lisziewicz et al., (2012) PLoS ONE 7:e35416) wasparticularly preferred. Furthermore, while non-viral vectors are muchsafer than viral vectors (i.e. low/nearly zero integration rates,lowered immunogenic potential), there is still a concern regardingimmunostimulatory prokaryotic CpG motifs in the backbone of traditionalplasmids. This concern is alleviated in MCs and/or nanoplasmids sincethese vectors lack a prokaryotic backbone or have a small bacterialregion size (less than 500 bp). SEAP was selected since it is of humanorigin so it should not cause an immunogenic reaction (Wang et al.,(2001) Gene 279: 99-108), and has already shown promise in the clinic(Kemp et al., (2008) Vaccine 26: 3608-3616).

Previously, viral infection has been used to drive cancer-specific geneconstructs, such as MC-OriP-IFNy (Zuo et al., (2011) PLoS ONE 6: e19407)which uses the viral OriP promoter/origin of replication to driveinterferon-y expression in Epstein-Barr virus (EBV) infectednasopharyngeal carcinomas (NPC). In contrast, the MC systems of thedisclosure can be broadly applicable for many different tumor typesbeyond viral-infected cells. The non-viral MC vectors of the presentdisclosure were developed for use in cancer screening using ablood-based assay.

Although tumor-activatable reporter gene-expressing vectors for cancerdetection have been developed (Bhang et al., (2011) Nat. Med. 17:123-129; Chaudhuri et al., (2003) Technol. In Cancer Res. & Treat. 2:171-180; Warram et al., (2011) Mol. Imaging Biol. 13: 452-461; Warram etal., (2012) Cancer Gene Therapy 19: 545-552; Browne et al., (2011) PLoSONE 6: e19530). The vector systems used in these cases (adenoviruses,Herpes simplex viruses, and plasmids); however, have safety issues thathamper clinical translation. Viruses are highly immunogenic andpre-existing viral immunity in humans is a widespread problem (Browne etal., (2011) PLoS ONE 6: e19530; Sumida et al., (2005) J. Immunol. 174:7179-7185; Schirmbeck et al., (2008) Mol. Therapy 16: 1609-1616).Plasmids can be immunogenic due to unmethylated CpG sequences in theprokaryotic backbone (necessary only for plasmid production) (Tan etal., (1999) Human Gene Therapy 10: 2153-2161), as well as typicallybearing coded antibiotic resistance genes to endogenous flora (Marie etal., (2010) J. Gene Med. 12: 323-332). Thus, the tumor-activatable MCsof the present disclosure have advantages over these other vectors andoffer translational potential primarily due to easier manufacturingpractices (compared to viruses) and a more desirable profile.

The MC and/or nanoplasmid systems of the disclosure can provide improvedspecificity through two mechanisms: 1) the uniqueness of the biomarkerin the blood since no SEAP is detectable prior to MC administration; and2) the ability to drive expression strictly within the tumor, therebyalleviating signal in healthy tumor-free subjects. A slight SEAP signalfrom tumor-free mice receiving MC likely is from leakiness of pSurv. Itis contemplated, however, that the MC systems of the disclosure are notlimited to this particular promoter and alternative tumor-activatablepromoters such as, but not limited to, the Idl or hTERT promoters(Warram et al., (2011) Mol. Imaging Biol. 13: 452-461; Zhang et al.,(2008) Life sciences 82: 1154-1161) and the like are useful in the MCsof the disclosure. Also, sensitivity using endogenous biomarkers isinherently limited by the amount of biomarker produced by the tumor(Hori & Gambhir (2011) Sci. Translational Med. 3: 109ra116). Incontrast, the sensitivity of the MC systems of the disclosure can bemodified.

One of the advantages of endogenous blood biomarkers is that they can beused to determine what type of cancer a person may harbor (e.g. a highPSA level may indicate prostate cancer). However, the MC systemsprovided by the present disclosure are also advantageous for screeningfor all cancer, not a particular tumor type. It is further contemplatedthat alternative promoters useful for screening patients at high-riskfor a particular cancer, such as variants of the prostate-specificantigen enhancer/promoter for prostate cancer (Iyer et al., (2005)Transgenic Res. 14: 47-55; Iyer et al., (2004) Mol. Therapy 10: 545-552;Iyer et al., (2006) Human Gene Therapy 17: 125-132) or the mucin-1promoter for breast cancer (Huyn et al., (2009) Clin. Cancer Res. 15:3126-3134) and the like can be incorporated into the MC systems of thedisclosure.

Another limitation of exogenous biomarkers (i.e. reporter) is theinability to localize the site(s) in the body where the biomarkeroriginated. By replacing or co-expressing SEAP with an imaging reportergene (e.g., herpes simplex virus thymidine kinase 1 for positronemission tomography (PET), which is described in e.g. Yaghoubi S S andGambhir S S (2006) Nat Protoc. 1(6):3069-75.) the systems of thedisclosure can also allow tumor location to be visualized. Bhang et al.recently described the ability to image tumors using both BLI and singlephoton emission computed tomography (SPECT) following systemicadministration of tumor-activatable plasmids expressing the appropriateimaging reporter gene (Bhang et al., (2011) Nat. Med. 17: 123-129). Thisstrategy was also pursued with the SEAP-expressing viral vectorsdescribed to date since these vectors co-expressed fluorescent proteinsfor cancer visualization using fluorescence stereomicroscopy (Chaudhuriet al., (2003) Technol. In Cancer Res. & Treat. 2: 171-180; Warram etal., (2011) Mol. Imaging Biol. 13: 452-461; Warram et al., (2012) CancerGene Therapy 19: 545-552). Rather than one vector system expressing tworeporters. It is further contemplated to be possible to deliver twodifferent vectors designed for specific applications; one for cancerscreening expressing a secretable reporter, and one for tumorlocalization expressing an imaging reporter.

One aspect of the disclosure, therefore, encompasses embodiments of arecombinant nucleic acid minicircle vector comprising a nucleotidesequence operably linked to a tumor-specific gene expression promoterand results in expression at a level greater by a recipient tumor cellthan by a non-tumor cell.

In the embodiments of this aspect of the disclosure the tumor-specificgene expression promoter may be selected from the group consisting of:Survivin promoter (BIRC5), a CXCR4 promoter, an ATP binding cassettesubfamily C member 4 (ABCC4) promoter, an anterior gradient 2, proteindisulphide isomerase family member (AGR2) promoter, activation inducedcytidine deaminase (AICDA) promoter, an UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin3 (CDH3) promoter, a CEA cell adhesion molecule 5 (CEACAM5) promoter, acentromere protein F (CENPF) promoter, a centrosomal protein 55 (CEP55)promoter, a claudin 3 (CLDN3) promoter, a claudin 4 (CLDN4) promoter, acollagen type XI alpha 1 chain (COL11A1) promoter, a collagen type Ialpha 1 chain (COL1A1) promoter, a cystatin SN (CST1) promoter, adenticleless E3 ubiquitin protein ligase homolog (DTL) promoter, afamily with sequence similarity 111 member B (FAM111B) promoter, aforkhead box A1 (FOXA1) promoter, a kinesin family member 20A (KIF20A),a laminin subunit gamma 2 (LAMC2) promoter, a mitotic spindlepositioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group BD (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof.

In some embodiments of this aspect of the disclosure, the nucleotidesequence operably linked to the tumor-specific promoter can be expressedas a polypeptide.

In some embodiments of this aspect of the disclosure, the nucleotidesequence operably linked to the tumor-specific promoter can encode areporter polypeptide.

In some embodiments of this aspect of the disclosure, the reporterpolypeptide may be an MRI reporter, a PET reporter; a SPECT reporter, aphotoacoustic reporter, a bioluminescent reporter, or any combinationthereof.

In some embodiments of this aspect of the disclosure, the polypeptidecan be secreted embryonic alkaline phosphatase (SEAP).

In some embodiments of this aspect of the disclosure, the recombinantnucleic acid minicircle vector can have the nucleic acid sequenceaccording to SEQ ID NO: 1.

In some embodiments of this aspect of the disclosure, the polypeptidecan be a bioluminescent reporter.

In some embodiments of this aspect of the disclosure, the recombinantnucleic acid minicircle vector can have the nucleic acid sequenceaccording to SEQ ID NO: 2.

In some embodiments of this aspect of the disclosure, the nucleotidesequence operably linked to the tumor-specific promoter can be expressedas a small interfering RNA (siRNA) or a therapeutically effectivepolypeptide.

Another aspect of the disclosure encompasses embodiments of apharmaceutically acceptable composition comprising a recombinant nucleicacid minicircle vector comprising a nucleotide sequence operably linkedto a tumor-specific gene expression promoter and expressible at a levelgreater by a recipient tumor cell than by a non-tumor cell, and apharmaceutically acceptable carrier, wherein: (i) the tumor-specificgene expression promoter can be selected from the group consisting of: aSurvivin promoter (BIRC5), a CXCR4 promoter, an ATP binding cassettesubfamily C member 4 (ABCC4) promoter, an anterior gradient 2, proteindisulphide isomerase family member (AGR2) promoter, activation inducedcytidine deaminase (AICDA) promoter, an UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin3 (CDH3) promoter, a CEA cell adhesion molecule 5 (CEACAM5) promoter, acentromere protein F (CENPF) promoter, a centrosomal protein 55 (CEP55)promoter, a claudin 3 (CLDN3) promoter, a claudin 4 (CLDN4) promoter, acollagen type XI alpha 1 chain (COL11A1) promoter, a collagen type Ialpha 1 chain (COL1A1) promoter, a cystatin SN (CST1) promoter, adenticleless E3 ubiquitin protein ligase homolog (DTL) promoter, afamily with sequence similarity 111 member B (FAM111B) promoter, aforkhead box A1 (FOXA1) promoter, a kinesin family member 20A (KIF20A),a laminin subunit gamma 2 (LAMC2) promoter, a mitotic spindlepositioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group IID (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof and (ii) the nucleotide sequence operably linkedto the tumor-specific promoter can be expressed as a polypeptideencoding an MRI reporter, a PET reporter, a SPECT reporter, aphotoacoustic reporter, a bioluminescent reporter, or any combinationthereof.

In some embodiments of this aspect of the disclosure, the recombinantnucleic acid minicircle vector can have the nucleic acid sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 2.

Yet another aspect of the disclosure encompasses embodiments of a methodof detecting a tumor cell in a human or non-human subject, comprisingthe steps of: (i) delivering to a first subject human or non-humananimal a pharmaceutically acceptable composition comprising arecombinant nucleic acid minicircle vector comprising a nucleotidesequence operably linked to a tumor-specific gene expression promoterand expressible at a level greater by a recipient tumor cell than by anon-tumor cell, and a pharmaceutically acceptable carrier, wherein: (a)the tumor-specific gene expression promoter can be selected from thegroup consisting of: Survivin promoter (BIRC5), a CXCR4 promoter, an ATPbinding cassette subfamily C member 4 (ABCC4) promoter, an anteriorgradient 2, protein disulphide isomerase family member (AGR2) promoter,activation induced cytidine deaminase (AICDA) promoter, anUDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3)promoter, a cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5(CEACAM5) promoter, a centromere protein F (CENPF) promoter, acentrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3) promoter, aclaudin 4 (CLDN4) promoter, a collagen type XI alpha 1 chain (COL11A1)promoter, a collagen type I alpha 1 chain (COL1A1) promoter, a cystatinSN (CST1) promoter, a denticleless E3 ubiquitin protein ligase homolog(DTL) promoter, a family with sequence similarity 111 member B (FAM111B)promoter, a forkhead box A1 (FOXA1) promoter, a kinesin family member20A (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitoticspindle positioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group BD (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof and (b) the nucleotide sequence operably linkedto the tumor-specific promoter can be expressed as a polypeptideencoding an MRI reporter, a PET reporter, a SPECT reporter, aphotoacoustic reporter, a bioluminescent reporter, or any combinationthereof; and (ii) detecting an expression product in the first subject,wherein said expression product is generated from the nucleotidesequence operably linked to the tumor-specific gene expression promoterof the minicircle vector, and wherein the detection of said expressionproduct indicates the presence of a tumor cell in the first subject.

In some embodiments of this aspect of the disclosure, the expressionproduct can be a serum polypeptide and step (ii) can comprise obtaininga serum sample from the first subject and determining the serum level ofthe expression product generated from the minicircle vector.

In some embodiments of this aspect of the disclosure, the detectedexpression product can be secreted embryonic alkaline phosphatase(SEAP).

In some embodiments of this aspect of the disclosure, the minicirclevector can have the nucleic acid sequence according to SEQ ID NO: 1.

In some embodiments of this aspect of the disclosure, the expressionproduct can be a bioluminescent polypeptide and the step (ii) cancomprise generating a detectable signal derived from the expressionproduct, measuring the level of the detectable signal generated from theminicircle vector, and comparing the level of the signal from the firstsubject to that obtained from a second subject not receiving theminicircle vector, wherein an elevated level signal from the firstsubject compared to that level obtained from a second subject indicatesthat the first subject comprises a tumor cell or population of tumorcells.

In some embodiments of this aspect of the disclosure, the step (ii) canfurther comprise non-invasively detecting the detectable signal,converting said signal into an image, overlaying said image with animage of the first subject, and locating the detectable signal relativeto the first subject, thereby determining the position of a tumor cellor population of tumor cells in the first subject.

In some embodiments of this aspect of the disclosure, the expressionproduct can be a luciferase.

In some embodiments of this aspect of the disclosure, the minicirclevector can have the nucleic acid sequence according to SEQ ID NO: 2.

Improved Synthetic Biomarkers for Disease Diagnosis, Detection, andMonitoring

In some aspects, the present disclosure provides for a methodcomprising: (a) administering to a subject a composition, wherein thecomposition induces expression of a synthetic biomarker in a diseasedcell preferentially over expression of the biomarker in non-diseasedcells in the subject such that a relative concentration ratio of thebiomarker expressed in the diseased cell over the non-diseased cells isgreater than about 1.0; (b) detecting the synthetic biomarker; and (c)using the synthetic biomarker detected in (b) to detect that the subjecthas the diseased cell. In some embodiments, the detecting has anaccuracy of at least 90%.

In some cases, the composition is administered intravenously,subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally, byinhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-17). In someembodiments, the composition is administered into at least one of thecervical, epitrochlear, supraclavicular, cervical, axillary,mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteallymph nodes. In some cases, lymph-node based administration may serve asa method of centralized local delivery to a tissue region.

In some cases, the detection of the diseased cell may have an accuracyat least about 50%, at least about 53%, at least about 55%, at leastabout 57%, at least about 60%, at least about 63%, at least about 65%,at least about 67%, at least about 70%, at least about 72%, at leastabout 75%, at least about 77%, at least about 78%, at least about 79%,at least about 80%, at least about 81%, at least about 82%, 83%, atleast about 84%, 85%, at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, or any range in between these values. Insome cases the detection of the diseased cell may have an accuracy of atmost about 53%, 55%, 57%, 60%, 63%, 65%, 67%, 70%, 72%, 75%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, or any range in between these values.

In some cases, the detection of the diseased cell may have a sensitivityof at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range in betweenthese values. In some cases, the detection of the diseased cell may havea sensitivity of at most about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any rangein between these values.

In some cases, the detection of the diseased cell may have a specificityof at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range in betweenthese values. In some cases, the detection of the diseased cell may havea specificity of at most about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any rangein between these values

In some cases, the detection of the diseased cell may have a negativepredictive value (NPV) of at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.2%, 95.5%, 95.7%,96%, 96.2%, 96.5%, 96.7%, 97%, 97.2%, 97.5%, 97.7%, 98%, 98.2%, 98.5%,98.7%, 99%, 99.2%, 99.5%, 99.7%, or 99.9%, or any range in between thesevalues. In some cases, the detection of the diseased cell may have a NPVof at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 95.2%, 95.5%, 95.7%, 96%, 96.2%, 96.5%, 96.7%, 97%,97.2%, 97.5%, 97.7%, 98%, 98.2%, 98.5%, 98.7%, 99%, 99.2%, 99.5%, 99.7%,or 99.9%, or any range in between these values.

In some cases, the detection of the diseased cell may have a positivepredictive value (PPV) of at least about 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 63%, 65%, 67%, 70%, 72%,75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any rangebetween these values. In some cases, the detection of the diseased cellmay have a PPV of at most about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 60%, 63%, 65%, 67%, 70%, 72%, 75%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range between these values.

In some embodiments, the composition may comprise a vector encoding thesynthetic biomarker. Suitable vectors include vectors suitable foradministration to cells in vivo, including but not limited tominicircles, plasmids, nanoplasmids, mini-intronic plasmids, yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),cosmids, phagemids, bacteriophages, and baculoviruses. Suitable vectorsalso include vectors derived from bacteriophages or plant, invertebrate,or animal (including human) viruses such as CELiD vectors,adeno-associated viral vectors (e.g. AAV1, AAV2, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, or pseudotyped combinations thereof such as AAV2/5, AAV2/2,AAV-DJ, or AAV-DJ8), retroviral vectors (e.g. MLV or self-inactivatingor SIN versions thereof, or pseudotyped versions thereof), herpesvirus(e.g. HSV- or EBV-based), lentivirus vectors (e.g. HIV-, FIV-, orEIAV-based, or pseudotyped versions thereof), or adenoviral vectors(e.g. Ad5-based, including replication-deficient, replication-competent,or helper-dependent versions thereof). In some cases, the vector maycomprise an episomal maintenance element to facilitate replication inone or more target cell type, such as a Scaffold/Matrix AttachmentRegion (S/MAR). S/MAR elements are particularly useful to facilitatereplication in the context of “naked” nucleic acid vectors such asminicircles. Exemplary suitable S/MAR elements include, but are notlimited to, EμMAR from the immunoglobulin heavy chain locus, the apoBMAR from the human apolipoprotein B locus, the Ch-LysMAR from thechicken lysozyme locus, and the huIFNβ MAR from the human IFNβ-locus. Insome embodiments, the vector may be a non-viral vector.

In some cases, the composition may comprise a vector containing asequence encoding the synthetic biomarker operably linked to a promoter.Suitable promoters include natural pan-tumor specific promoters, naturaltissue specific promoters, natural disease-specific/disease-activatedpromoters, natural constitutive promoters, and any composites thereof.The promoter may be a Survivin promoter (BIRC5), a CXCR4 promoter, anATP binding cassette subfamily C member 4 (ABCC4) promoter, an anteriorgradient 2, protein disulphide isomerase family member (AGR2) promoter,activation induced cytidine deaminase (AICDA) promoter, anUDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3)promoter, a cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5(CEACAM5) promoter, a centromere protein F (CENPF) promoter, acentrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3) promoter, aclaudin 4 (CLDN4) promoter, a collagen type XI alpha 1 chain (COL11A1)promoter, a collagen type I alpha 1 chain (COL1A1) promoter, a cystatinSN (CST1) promoter, a denticleless E3 ubiquitin protein ligase homolog(DTL) promoter, a family with sequence similarity 111 member B (FAM111B)promoter, a forkhead box A1 (FOXA1) promoter, a kinesin family member20A (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitoticspindle positioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group BD (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof.

In some cases, the synthetic biomarker may be a polypeptide or nucleicacid biomarker. Polypeptides include any of the reporter polypeptidesdescribed herein. Nucleic acids include natural or engineered miRNAs,RNA hairpins, and RNA aptamers or barcoded versions thereof. When thenucleic acid is an miRNA, the miRNA may be detected e.g. by standardlibrary generation techniques such as degenerate primer-based annealingand ligation, poly(A) polymerase labeling followed by RT or ligation, orsequential adapter ligation coupled to q-PCR, sequencing, or anelectrophoretic detection method. When the biomarker is a polypeptide,the polypeptide may comprise an N-terminal secretion signal sequence(e.g. the N-terminal signal peptide from CD33 or CD8a).

By ascribing an exclusive label to a unique member within a largergroup, barcodes afford the opportunity to identify and quantify thatmember (e.g. expression of a reporter under the control of a particularcancer specific promoter) within the context of a larger and morecomplex mixture of many members (e.g. multiple promoter-reporterconstructs expressed within the same cell), as well as offering theopportunity to isolate a single member from the complex mixture. Forinstance, in the case of barcodes based on nucleic acids, hybridizationof barcodes based on base pairing complementarity may be used to captureand isolate or otherwise reduce the complexity of a mixture by saidcapture event. For barcodes based on peptides, unique features includingimmunocapture or interactions of ligands and receptors may be used tocapture and isolate or otherwise reduce the complexity of a mixture bysaid capture event.

When the nucleic acid is an engineered miRNA, the nucleic acid may bethe Sec-miR or miR-neg constructs described in Ronald et al. (Ronald etal. PLoS ONE 11(7): e0159369.) Such constructs comprise: (a) a codingsequence not expressed endogenously and not having any known vertebratetarget (e.g. Sec-miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) miR backbonesequences providing processing of pre-miRNA to mature miRNA flanking thecoding sequence (e.g. miR-155 or miR-130 backbone sequences); and (c) anEXOmotif enhancing loading into exosomes (e.g. GGAG). Such miRNAconstructs may be expressed in e.g. the 3′-UTR of a gene encoding areporter polypeptide, or from the 3′-UTR of a gene encoding a suitablynon-toxic protein (e.g. an endogenous structural protein such as actinor tubulin, or a highly expressed protein such as ubiquitin). In someembodiments, multiple copies (e.g. at least 2, at least 4) of theengineered miRNA may be provided in tandem.

In some cases, the synthetic biomarker may be a polypeptide biomarkerdetectable by a non-invasive imaging method performed on the subjectand/or the method comprises detecting the synthetic biomarker bynon-invasive imaging. Such non-invasive imagine methods include MRIimaging, PET imaging, SPECT imaging, photoacoustic imaging, andbioluminescent imaging. Synthetic biomarkers detectable by MRI imaginginclude polypeptide contrast agents, such as ferritin (or mutantsthereof, such as Pyrococcus furiousus ferritin mutants L55P, F57S, orF123S), or lanthanide-binding proteins (or engineered fusions thereof,such as the LBT-ubiquitin fusions described in Daughtry et al.ChemBioChem 2012, 13, 2567-2574). Synthetic biomarkers detectable by PETor SPECT imaging include the human sodium iodide symporter (e.g. inconjunction with administration of PET-active iodine/iodide isotopes,see e.g. Penheiter et al. Curr Gene Ther. 2012 February; 12(1): 33-47),HSV-tk or mutants thereof such as HSV-sr39tk (e.g. in conjunction withadministration of positron-labeled acycloguanosine or pyrimidine analogPET reporters such as [18F]FHBG, see Yaghoubi S S et al. Nat Protoc.2006; 1(6):3069-75), and the dopamine D2 receptor or mutants thereofsuch as D2R80A or D2R194A (e.g. in conjunction with administration ofpositron-labeled D2 binders such as 3-(2′[18F]-fluoroethyl)-spiperone).Synthetic biomarkers detectable by photoacoustic imaging include thepigment-producing enzymes such as β-galactosidase (e.g. in combinationwith administration of X-gal) and tyrosinase, autofluorescent proteins(e.g. GFP, mCherry, or derivatives thereof), non-fluorescent GFP-likechromoproteins (e.g. aeCP597 and cjBlue and derivatives thereof),bacteriophytochrome-based near-infrared fluorescent proteins (e.g.IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713/V256C,iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphP1-FP/C20S, orAphB variants), and reversibly photoswitchable proteins (e.g. Dronpa,Dronpa-M159T, and BphP1 or variants thereof). Synthetic biomarkersdetectable by bioluminescent imaging include luciferases (e.g. incombination with administration of coelenterazines described herein),including Gaussia luciferases, Renilla luciferases, and Photinusluciferases (e.g. including the engineered Ppy RE8 and RE9 versionsdescribed in Branchini et al. Anal. Biochem. 396(2010): 290-297). Insome embodiments, the synthetic biomarker may be a contrast agent, anenzyme producing a detectable molecule, or a transporter drivingaccumulation of a detectable molecule. The synthetic biomarker may bemeasured in situ within subject's body.

In instances where the synthetic biomarker is a polypeptide biomarkerdetectable by a non-invasive imaging method, the method involvingadministering to a subject a composition inducing expression of asynthetic biomarker in a diseased cell may further comprise (d)localizing the diseased cell in the body of the subject. The localizingmay be associated with a particular resolution, for example 10 mm to 10cm, at least 10 mm, or at most 10 cm. The localizing may be associatedwith a particular minimum detectable tumor size, for example a tumorsize between 3 mm³ and 5 cm³. In some cases, the particular minimumrange may be 1 cm³ to 5 cm³, or 900 mm³ to 1 cm³, or 800 mm³ to 900 mm³,or 700 mm³ to 800 mm³, or 600 mm³ to 700 mm³, or 500 mm³ to 600 mm³, or400 mm³ to 500 mm³, or 300 mm³ to 400 mm³, or 200 mm³ to 300 mm³, or 100mm³ to 200 mm³, or 50 mm³ to 100 mm³, or 10 mm³ to 50 mm³, or 3 mm³ to10 mm³ in size. In some cases, the localization occurs in a non-invasiveimaging scan (e.g. PET, MRI, SPECT, etc). In some cases, thelocalization occurs during surgical intervention in situ, for example bythe use of visual inspection (in the case of visual-range absorbingreporters) or by the use of visual inspection combined with fluorescentexcitation.

In some cases, the additional localization step above may be followed bya surgical step to eliminate the detected and/or localized diseasedcell. The surgical step may be performed by the same or different partyto that which administers the biomarker-encoding composition and/orlocalizes the diseased cell. The surgical step may be surgical excisionof the diseased cell or a tumor associated with the diseased cell. Thesurgical or nonsurgical elimination step may involve aminimally-invasive killing technique, such as a radiosurgery (includingbut not limited to Gamma Knife, Reflexion, CyberKnife, and relatedtechniques using targeted ionizing radiation to kill diseased cells).

In some cases, the synthetic biomarker may be detected in biologicalsample from the subject to whom the composition inducing expression ofthe synthetic biomarker is administered. In some cases, the syntheticbiomarker is detected in vivo and determines a location of the diseasedcell.

In some cases, the composition administered to the subject may comprisea transfection agent. Suitable transfection agents include, but are notlimited to, linear or branched polyethylenimines, nanoparticles,lipophilic particles, peptides, micelles, dendrimers, hydrogels,synthetic or naturally derived exosomes, polymeric composition,virus-like particles, and any combination thereof.

In some cases, the composition may further comprise a pharmaceuticallyacceptable carrier. Exemplary pharmaceutically acceptable carriersinclude, but are not limited to, water, peanut oil, soybean oil, mineraloil, sesame oil, saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, aqueous dextrose, glycerol solution,glucose, lactose, sucrose, glycerol monostearate, sodium chloridesolution, propylene, glycol, or ethanol, or any combination thereof.

The biological sample may be a sample collected by a non-invasive methodfrom the subject. Exemplary non-invasive samples include, but are notlimited to, saliva, sputum, sweat, urine, stool, semen, cervicovaginalsecretions, breast milk, rheum, tears, and cheek epithelial swabs. Thebiological sample may be a sample collected by a minimally-invasivemethod from the subject. Exemplary minimally-invasive samples include,but are not limited to, blood samples (e.g. obtained by venipuncture orcapillary tube), pleural fluid samples (e.g. obtained by thoracentesis),amniotic fluid samples (e.g. obtained by amniocentesis), and gastricfluid samples (e.g. obtained by gastric lavage). The biological samplemay be a sample obtained by biopsy, such as a skin biopsy sample (e.g.obtained by punch, shave, saucerization, wedge, incisional, orexcisional biopsy), a bone marrow sample (e.g. obtained by aspirationbiopsy), a lymph node or breast biopsy (e.g. obtained by fine-needleaspiration, core needle biopsy, vacuum assisted biopsy, or image-guidedbiopsy), a surgical biopsy sample (e.g. of an internal organ obtained byexcisional or incisional biopsy), or a mouth, GI-tract, lung, bladder,or urinary tract biopsy (e.g. obtained by endoscopy).

In some cases, the biological sample may be obtained a certain period oftime after administration of the composition inducing expression of thesynthetic biomarker. The biological sample may be obtained at leastabout 15 minutes, at least about 30 minutes, at least about 1 hour, atleast about 2 hours, at least about 4 hours, at least about 8 hours, atleast about 16 hours, at least about 24 hours, at least about 36 hours,at least about 48 hours, at least about 3 days, at least about 4 days,at least about 5 days, at least about 6 days, at least about 7 days, atleast about 8 days, at least about 9 days, at least about 10 days, atleast about 11 days, at least about 12 days, at least about 13 days, atleast about 14 days, at least about 15 days, at least about 1 month, atleast about 2 months, at least about 3 months, at least about 4 months,at least about 5 months, or at least about 6 months after administrationof the composition inducing expression of the synthetic biomarker. Thebiological sample may be obtained at most about 15 minutes, at mostabout 30 minutes, at most about 1 hour, at most about 2 hours, at mostabout 4 hours, at most about 8 hours, at most about 16 hours, at mostabout 24 hours, at most about 36 hours, at most about 48 hours, at mostabout 3 days, at most about 4 days, at most about 5 days, at most about6 days, at most about 7 days, at most about 8 days, at most about 9days, at most about 10 days, at most about 11 days, at most about 12days, at most about 13 days, at most about 14 days, at most about 15days, at most about 1 month, at most about 2 months, at most about 3months, at most about 4 months, at most about 5 months, or at most about6 months after administration of the composition inducing expression ofthe synthetic biomarker. In some embodiments, the biological sample maybe obtained, and any biomarker detection protocols performed multipletimes post administration of the composition inducing expression of thesynthetic biomarker (e.g. to monitor synthetic biomarker levels overtime). The biological sample may be obtained at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, or 25 times post administration of thecomposition inducing expression of the synthetic biomarker. Thebiological sample may be obtained weekly or monthly followingadministration of the composition inducing expression of the syntheticbiomarker.

In some cases, the diseased cell may be a cancerous cell, a cellindicative of an autoimmune disease (e.g. a T-cell or lymphocyte withself-directed activity, or a normal cell damaged by autoimmunity), acell indicative of a neurodegenerative disease (e.g. a cell bearing atoxic amyloid or proximal to a toxic amyloid), or a cell that may havean altered gene expression profile because a subject from which the cellis obtained suffers a disease or is about to suffer from a disease. Acell population comprising cells that have an altered gene expressionprofile can be described as transcriptionally altered cells (TACs). Insome cases, the diseased cell may be a cancerous cell. Exemplary cancersinclude, but are not limited to, carcinomas, sarcomas, lymphomas,leukemias, and adenomas. Carcinomas may arise from cells that coverinternal and external parts of the body such as the lung, breast, andcolon. Sarcomas may arise from cells that are located in bone,cartilage, fat, connective tissue, muscle, and other supportive tissues.Lymphomas may arise in the lymph nodes and immune system tissues.Leukemias may arise in the bone marrow and accumulate in thebloodstream. Adenomas may arise in the thyroid, the pituitary gland, theadrenal gland, and other glandular tissues. Specific exemplary examplesof cancer types include suitable for detection with the methodsaccording to the disclosure include acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basalcell carcinoma, bile duct cancer, bladder cancer, bone cancers, braintumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignantglioma, ependymoma, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, breastcancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknownprimary origin, central nervous system lymphoma, cerebellar astrocytoma,cervical cancer, childhood cancers, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic myeloproliferative disorders,colon cancer, cutaneous T-cell lymphoma, desmoplastic small round celltumor, endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma, germ cell tumors, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor,gliomas, hairy cell leukemia, head and neck cancer, heart cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer,intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidneycancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, livercancer, lung cancers, such as non-small cell and small cell lung cancer,lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytomaof bone/osteosarcoma, medulloblastoma, melanomas, mesothelioma,metastatic squamous neck cancer with occult primary, mouth cancer,multiple endocrine neoplasia syndrome, myelodysplastic syndromes,myeloid leukemia, nasal cavity and paranasal sinus cancer,nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-smallcell lung cancer, oral cancer, oropharyngeal cancer,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer,ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer,pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonaryblastoma, plasma cell neoplasia, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renalpelvis and ureter transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skincarcinoma merkel cell, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer,thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor(gestational), cancers of unknown primary site, urethral cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia,and Wilms tumor.

In some cases, the diseased cell may be a virally-infected cell.Exemplary viruses include, but are not limited to, HIV, hepatitis Cvirus, hepatitis B virus, hepatitis D virus, herpesviruses, Epstein-Barrvirus, cytomegalovirus, and human T-lymphotropic virus type III.

In some cases, the diseased cell may be indicative of an autoimmunedisease. Exemplary autoimmune diseases include, but are not limited to,Achalasia, Addison's disease, Adult Still's disease, Agammaglobulinemia,Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmunedysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis,Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmuneoophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmuneretinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN),Baló disease, Behcet's disease, Benign mucosal pemphigoid, Bullouspemphigoid, Castleman disease (CD), Celiac disease, Chagas disease,Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronicrecurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS)or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan'ssyndrome, Cold agglutinin disease, Congenital heart block, Coxsackiemyocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis,Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus,Dressler's syndrome, Endometriosis, Eosinophilic esophagitis (EoE),Eosinophilic fasciitis, Erythema nodosum, Essential mixedcryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis,Giant cell arteritis (temporal arteritis), Giant cell myocarditis,Glomerulonephritis, Goodpasture's syndrome, Granulomatosis withPolyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto'sthyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpesgestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa(HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy,IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP),Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenilearthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM),Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis,Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgAdisease (LAD), Lupus, Lyme disease chronic, Meniere's disease,Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy(MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis,Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocularcicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR),PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis(peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheralneuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMSsyndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III,Polymyalgia rheumatica, Polymyositis, Postmyocardial infarctionsyndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis,Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis,Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum,Raynaud's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy,Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitonealfibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidtsyndrome, Scleritis, Scleroderma, Sjögren's syndrome, Sperm & testicularautoimmunity, Stiff person syndrome (SPS), Subacute bacterialendocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transversemyelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiatedconnective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, andVogt-Koyanagi-Harada Disease.

In some cases, the diseased cell may be indicative of aneurodegenerative disease. Neurodegenerative diseases include, but arenot limited to, Multiple sclerosis (MS), Alzheimer's disease (AD),Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), orneurodegeneration due to infection by viruses of families Herpesviridae,Polyomaviridae, Bornaviridae, Orthomyxoviridae, Paramyxoviridae,Rhabdoviridae, Flaviviridae, Picornaviridae, or Retroviridae (see Zhouet al. Virol J. 2013; 10: 172).

Genetic/DNA-Based Therapeutics for Diseased Cells

In some aspects, the present disclosure provides for a method oftreating a subject having or suspected of having a disease, comprisingadministering to the subject a composition that induces expression of atherapeutically effective agent by a diseased cell associated with thedisease preferentially over expression of the therapeutically effectiveagent by non-diseased cells in the subject such that a relativeconcentration of the therapeutically effective agent expressed by thediseased cell over the non-diseased cells is greater than 1.0, whichtherapeutically effective agent treats the subject at a therapeuticefficacy of at least 10% as determined by a decrease in a cellpopulation of the diseased cell.

In some cases, the composition is administered intravenously,subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally,inhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-175). In someembodiments, the composition is administered into at least one of thecervical, epitrochlear, supraclavicular, cervical, axillary,mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteallymph nodes. In some cases, lymph-node based administration may serve asa method of centralized local delivery to a tissue region.

In some cases, the composition administered for treating a subjecthaving or suspected of having a disease may comprise a promoter operablylinked to a nucleotide sequence encoding the therapeutically effectiveagent. The promoter may be a cancer-specific promoter. Suitablepromoters include natural pan-tumor specific promoters, natural tissuespecific promoters, natural disease-specific/disease-activatedpromoters, natural constitutive promoters, and any composites thereof.The promoter may be a Survivin promoter (BIRC5), a CXCR4 promoter, anATP binding cassette subfamily C member 4 (ABCC4) promoter, an anteriorgradient 2, protein disulphide isomerase family member (AGR2) promoter,activation induced cytidine deaminase (AICDA) promoter, anUDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3)promoter, a cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5(CEACAM5) promoter, a centromere protein F (CENPF) promoter, acentrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3) promoter, aclaudin 4 (CLDN4) promoter, a collagen type XI alpha 1 chain (COL11A1)promoter, a collagen type I alpha 1 chain (COL1A1) promoter, a cystatinSN (CST1) promoter, a denticleless E3 ubiquitin protein ligase homolog(DTL) promoter, a family with sequence similarity 111 member B (FAM111B)promoter, a forkhead box A1 (FOXA1) promoter, a kinesin family member20A (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitoticspindle positioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group IID (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof.

In some cases, the promoter operably linked to a nucleotide sequenceencoding the therapeutically effective agent may be present on a vector,which may be a component of the composition administered to the subject.Suitable vectors include vectors suitable for administration to cells invivo, including but not limited to minicircles, plasmids, nanoplasmids,mini-intronic plasmids, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), cosmids, phagemids, bacteriophages, andbaculoviruses. Suitable vectors also include vectors derived frombacteriophages or plant, invertebrate, or animal (including human)viruses such as CELiD vectors, adeno-associated viral vectors (e.g.AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotypedcombinations thereof such as AAV2/5, AAV2/2, AAV-DJ, or AAV-DJ8),retroviral vectors (e.g. MLV or self-inactivating or SIN versionsthereof, or pseudotyped versions thereof), herpesvirus (e.g. HSV- orEBV-based), lentivirus vectors (e.g. HIV-, FIV-, or EIAV-based, orpseudotyped versions thereof), or adenoviral vectors (e.g. Ad5-based,including replication-deficient, replication-competent, orhelper-dependent versions thereof). In some cases, the vector maycomprise an episomal maintenance element to facilitate replication inone or more target cell type, such as a Scaffold/Matrix AttachmentRegion (S/MAR). S/MAR elements are particularly useful to facilitatereplication in the context of “naked” nucleic acid vectors such asminicircles. Exemplary suitable S/MAR elements include, but are notlimited to, EμMAR from the immunoglobulin heavy chain locus, the apoBMAR from the human apolipoprotein B locus, the Ch-LysMAR from thechicken lysozyme locus, and the huIFINβ MAR from the human IFNβ-locus.In some embodiments, the vector may be a non-viral vector.

In some cases, the therapeutically effective agent may comprise aparticular class of therapeutic. Exemplary classes of therapeuticssuitable for use according to methods of the disclosure include, but arenot limited to, therapeutically effective polypeptides (e.g. therapeuticantibodies, fragments, or derivatives thereof; cytokines; growthfactors; engineered or replacement metabolic/catabolic enzymes,engineered short peptide agonists or antagonists, or prodrug activatingenzymes), small activating RNAs (saRNAs), microRNAs (miRNAs), smallinterfering RNAs (siRNAs) or any combination thereof. In some cases, thetherapeutically effective agent may be a prodrug-activating enzyme.Exemplary prodrug-activating enzymes include, but are not limited to,HSVtk, cytosine deaminase, DT diaphorase, nitroreductase, guaninephosphoribosyl transferase, purine nucleoside phosphorylase, thymidinephorphorylase, carboxylesterase, folylpolyglutamyl synthetase,carboxypeptidase A1, carboxypeptidase G2, and cytochrome P-450. In caseswhere the therapeutically effective agent is a prodrug-activatingenzyme, the method may comprise an additional administration of the drugaccording to any of the routes described herein. When thetherapeutically effective agent is a polypeptide, the polypeptide maycomprise an N-terminal secretion signal sequence (e.g. the N-terminalsignal peptide from CD33 or CD8a).

Improved Synthetic Biomarker Constructs and Methods to Normalize AcrossIndividual Subjects' Transfection Rates

In some aspects, the present disclosure provides for a compositioncomprising a first nucleic acid sequence encoding a first polypeptide ornucleic acid biomarker and a second nucleic acid sequence encoding asecond polypeptide or second nucleic acid biomarker, wherein thecomposition is configured such that when the composition is in a cell:the second polypeptide or the second nucleic acid biomarker is expressedin an amount that reflects delivery of at least the first and the secondnucleic acids to the cell, and the first polypeptide or nucleic acidbiomarker is expressed differentially in a diseased cell versus anon-diseased cell. In some cases, (i) the cell induces expression of thefirst nucleic acid sequence in a diseased cell preferentially overexpression of the first nucleic acid sequence in non-diseased cells,wherein the first polypeptide is a detectable biomarker or a therapeuticagent; and (ii) the cell induces expression of the second nucleic acidsequence equally in diseased and in non-diseased cells and the secondnucleic acid sequence yields the second polypeptide that is not thedetectable biomarker or the therapeutic agent, such that a level ofexpression of the second polypeptide provides a control for assessingthe relative level of the nucleic acid sequences in the cell. In somecases, the first nucleic acid sequence encoding the first polypeptideand the second nucleic acid sequence encoding the second polypeptide maybe on independent genetic constructs. In some cases, in the compositionthe sequences comprising the first nucleic acid sequence encoding thefirst polypeptide and the second nucleic acid sequence encoding thesecond polypeptide may be on independent genetic constructs. In somecases, the vector comprises: (a) a first promoter operably linked to thefirst nucleic acid sequence, wherein the promoter induces expression ofthe first nucleic acid sequence in a diseased cell preferentially overexpression of the first nucleic acid sequence in non-diseased cells; and(b) a second promoter sequence that induces expression equally indiseased and in non-diseased cells and is operably linked to the secondnucleic acid.

In some cases, the first polypeptide may be both a detectable biomarkerand a therapeutic agent. In some cases, the first polypeptide is atherapeutic antibody, a therapeutic antibody fragment or derivative, ora prodrug-activating enzyme. Exemplary prodrug-activating enzymesinclude, but are not limited to, HSVtk, cytosine deaminase, DTdiaphorase, nitroreductase, guanine phosphoribosyl transferase, purinenucleoside phosphorylase, thymidine phorphorylase, carboxylesterase,folylpolyglutamyl synthetase, carboxypeptidase A1, carboxypeptidase G2,and cytochrome P-450. The polypeptide may comprise an N-terminalsecretion signal sequence (e.g. the N-terminal signal peptide from CD33or CD8a).

In some cases, the first and/or second nucleic acid may be on a vector.Suitable vectors include vectors suitable for administration to cells invivo, including but not limited to minicircles, plasmids, nanoplasmids,mini-intronic plasmids, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), cosmids, phagemids, bacteriophages, andbaculoviruses. Suitable vectors also include vectors derived frombacteriophages or plant, invertebrate, or animal (including human)viruses such as CELiD vectors, adeno-associated viral vectors (e.g.AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotypedcombinations thereof such as AAV2/5, AAV2/2, AAV-DJ, or AAV-DJ8),retroviral vectors (e.g. MLV or self-inactivating or SIN versionsthereof, or pseudotyped versions thereof), herpesvirus (e.g. HSV- orEBV-based), lentivirus vectors (e.g. HIV-, FIV-, or EIAV-based, orpseudotyped versions thereof), or adenoviral vectors (e.g. Ad5-based,including replication-deficient, replication-competent, orhelper-dependent versions thereof). In some cases, the vector maycomprise an episomal maintenance element to facilitate replication inone or more target cell type, such as a Scaffold/Matrix AttachmentRegion (S/MAR). S/MAR elements are particularly useful to facilitatereplication in the context of “naked” nucleic acid vectors such asminicircles. Exemplary suitable S/MAR elements include, but are notlimited to, EμMAR from the immunoglobulin heavy chain locus, the apoBMAR from the human apolipoprotein B locus, the Ch-LysMAR from thechicken lysozyme locus, and the huIFβ MAR from the human IFNβ-locus. Insome embodiments, the vector may be a non-viral vector.

In some cases, the cell to which the first and the second nucleic acidare delivered may be a diseased cell. In some cases, the diseased cellmay be a cancerous cell, a cell indicative of an autoimmune disease(e.g. a T-cell or lymphocyte with self-directed activity, or a normalcell damaged by autoimmunity), a TAC, or a cell indicative of aneurodegenerative disease (e.g. a cell bearing a toxic amyloid orproximal to a toxic amyloid). Exemplary cancers, autoimmune diseases,and neurodegenerative diseases which such a cell may be indicative ofinclude any of the cancers, autoimmune diseases, and neurodegenerativediseases described herein. In some cases, the diseased cell may be avirally-infected cell. Exemplary viruses include, but are not limitedto, HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus,herpesviruses, Epstein-Barr virus, cytomegalovirus, and humanT-lymphotropic virus type III.

In some cases, the first or the second nucleic acid may be detectablenucleic acid biomarkers. Exemplary detectable nucleic acids include, butare not limited to, natural or engineered miRNAs, RNA hairpins, and RNAaptamers or barcoded versions thereof. When the nucleic acid is anmiRNA, the miRNA may be detected e.g. by standard library generationtechniques such as degenerate primer-based annealing and ligation,poly(A) polymerase labeling followed by RT or ligation, or sequentialadapter ligation coupled to q-PCR, sequencing, or an electrophoreticdetection method. When the biomarker is a polypeptide, the polypeptidemay comprise an N-terminal secretion signal sequence (e.g. theN-terminal signal peptide from CD33 or CD8a).

When the nucleic acid is an engineered miRNA, the nucleic acid may bethe Sec-miR or miR-neg constructs described in Ronald et al. (Ronald etal. PLoS ONE 11(7): e0159369.) Such constructs comprise: (a) a codingsequence not expressed endogenously and not having any known vertebratetarget (e.g. Sec-miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) miR backbonesequences providing processing of pre-miRNA to mature miRNA flanking thecoding sequence (e.g. miR-155 or miR-130 backbone sequences); and (c) anEXOmotif enhancing loading into exosomes (e.g. GGAG). Such miRNAconstructs may be expressed in e.g. the 3′-UTR of a gene encoding areporter polypeptide, or from the 3′-UTR of a gene encoding a suitablynon-toxic protein (e.g. an endogenous structural protein such as actinor tubulin, or a highly expressed protein such as ubiquitin). In someembodiments, multiple copies (e.g. at least 2, at least 4) of theengineered miRNA may be provided in tandem.

In some cases, the second polypeptide or the first polypeptide may bedetectable by a non-invasive imaging method performed on the subject.Such non-invasive imagine methods include MRI imaging, PET imaging,SPECT imaging, photoacoustic imaging, and bioluminescent imaging.Synthetic biomarkers detectable by MRI imaging include polypeptidecontrast agents, such as ferritin (or mutants thereof, such asPyrococcus furiousus ferritin mutants L55P, F57S, or F123S), orlanthanide-binding proteins (or engineered fusions thereof, such as theLBT-ubiquitin fusions described in Daughtry et al. ChemBioChem 2012, 13,2567-2574). Synthetic biomarkers detectable by PET or SPECT imaginginclude the human sodium iodide symporter (e.g. in conjunction withadministration of PET-active iodine/iodide isotopes, see e.g. Penheiteret al. Curr Gene Ther. 2012 February; 12(1): 33-47), HSV-tk or mutantsthereof such as HSV-sr39tk (e.g. in conjunction with administration ofpositron-labeled acycloguanosine or pyrimidine analog PET reporters suchas [18F]FHBG, see Yaghoubi S S et al. Nat Protoc. 2006; 1(6):3069-75),and the dopamine D2 receptor or mutants thereof such as D2R80A orD2R194A (e.g. in conjunction with administration of positron-labeled D2binders such as 3-(2′[18F]-fluoroethyl)-spiperone). Synthetic biomarkersdetectable by photoacoustic imaging include the pigment-producingenzymes such as β-galactosidase (e.g. in combination with administrationof X-gal) and tyrosinase, autofluorescent proteins (e.g. GFP, mCherry,or derivatives thereof), non-fluorescent GFP-like chromoproteins (e.g.aeCP597 and cjBlue and derivatives thereof), bacteriophytochrome-basednear-infrared fluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev,IFP2.0, iRFP713, iRFP720, iRFP713/V256C, iRFP682, iRFP702, iRFP670,mIFP, iBlueberry, GAF-FP, BphP1-FP/C20S, or AphB variants), andreversibly photoswitchable proteins (e.g. Dronpa, Dronpa-M159T, andBphP1 or variants thereof). Synthetic biomarkers detectable bybioluminescent imaging include luciferases (e.g. in combination withadministration of coelenterazines described herein), including Gaussialuciferases, Renilla luciferases, and Photinus luciferases (e.g.including the engineered Ppy RE8 and RE9 versions described in Branchiniet al. Anal. Biochem. 396(2010): 290-297). In some embodiments, thesynthetic biomarker may be a contrast agent, an enzyme producing adetectable molecule, or a transporter driving accumulation of adetectable molecule. The synthetic biomarker may be measured in situwithin subject's body.

In some cases, the present disclosure provides for a method of detectingdiseased cells in a subject, comprising administering a composition tothe subject, wherein the composition comprises a first nucleic acidsequence encoding a first polypeptide or nucleic acid biomarker and asecond nucleic acid sequence encoding a second polypeptide or secondnucleic acid biomarker, wherein the composition is configured such thatwhen the composition is in a cell: the second polypeptide or the secondnucleic acid biomarker is expressed in an amount that reflects deliveryof at least the first and the second nucleic acids to the cell, and thefirst polypeptide or nucleic acid biomarker is expressed differentiallyin a diseased cell versus a non-diseased cell. In some cases, (i) thecell induces expression of the first nucleic acid sequence in a diseasedcell preferentially over expression of the first nucleic acid sequencein non-diseased cells, wherein the first polypeptide is a detectablebiomarker or a therapeutic agent; and (ii) the cell induces expressionof the second nucleic acid sequence equally in diseased and innon-diseased cells and the second nucleic acid sequence yields thesecond polypeptide that is not the detectable biomarker or thetherapeutic agent, such that a level of expression of the secondpolypeptide provides a control for assessing the relative level of thenucleic acid sequences in the cell. In some cases, the first nucleicacid sequence encoding the first polypeptide and the second nucleic acidsequence encoding the second polypeptide may be on independent geneticconstructs. In some cases, in the composition the sequences comprisingthe first nucleic acid sequence encoding the first polypeptide and thesecond nucleic acid sequence encoding the second polypeptide may be onindependent genetic constructs. In some cases, the vector comprises: (a)a first promoter operably linked to the first nucleic acid sequence,wherein the promoter induces expression of the first nucleic acidsequence in a diseased cell preferentially over expression of the firstnucleic acid sequence in non-diseased cells; and (b) a second promotersequence that induces expression equally in diseased and in non-diseasedcells and is operably linked to the second nucleic acid. In some cases,the method may comprise detecting the first polypeptide or nucleic acidbiomarker and/or the second polypeptide or nucleic acid biomarker. Insome cases, such method is a non-invasive imaging method performed onthe subject. Such non-invasive imagine methods include MRI imaging, PETimaging, SPECT imaging, photoacoustic imaging, and bioluminescentimaging.

In instances where the synthetic biomarker is a polypeptide biomarkerdetectable by a non-invasive imaging method, the method may furthercomprise localizing the diseased cell in the body of the subject. Thelocalizing may be associated with a particular resolution, for example10 mm to 10 cm, at least 10 mm, or at most 10 cm. The localizing may beassociated with a particular minimum detectable tumor size, for examplea tumor size between 3 mm3 and 10 cm³. In some cases, the particularminimum range may be may be 1 cm³ to 10 cm³, or 900 mm3 to 1 cm³, or 800mm³ to 900 mm³, or 700 mm³ to 800 mm³, or 600 mm³ to 700 mm³, or 500 mm³to 600 mm³, or 400 mm³ to 500 mm³, or 300 mm³ to 400 mm³, or 200 mm³ to300 mm³, or 100 mm³ to 200 mm³, or 50 mm³ to 100 mm³, or 10 mm³ to 50mm³, or 3 mm³ to 10 mm³ in size. In some cases, the localization occursin a non-invasive imaging scan (e.g. PET, MRI, SPECT, etc). In somecases, the localization occurs during surgical intervention in situ, forexample by the use of visual inspection (in the case of visual-rangeabsorbing reporters) or by the use of visual inspection combined withfluorescent excitation.

In some cases, the additional localization step above may be followed bya surgical step to eliminate the detected and/or localized diseasedcell. The surgical step may be performed by the same or different partyto that which administers the biomarker-encoding composition and/orlocalizes the diseased cell. The surgical step may be surgical excisionof the diseased cell or a tumor associated with the diseased cell. Thesurgical or nonsurgical elimination step may involve aminimally-invasive killing technique, such as a radiosurgery (includingbut not limited to Gamma Knife, Reflexion, CyberKnife, and relatedtechniques using targeted ionizing radiation to kill diseased cells).

In some cases, the non-invasive imaging method may be performed acertain period of time after administration of the composition inducingexpression of the synthetic biomarker. The non-invasive imaging methodmay be performed at least about 15 minutes, at least about 30 minutes,at least about 1 hour, at least about 2 hours, at least about 4 hours,at least about 8 hours, at least about 16 hours, at least about 24hours, at least about 36 hours, at least about 48 hours, at least about3 days, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 15days, at least about 1 month, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, or at least about 1 year after administration of thecomposition comprising the first and second nucleic acid. Thenon-invasive imaging method may be performed at most about 15 minutes,at most about 30 minutes, at most about 1 hour, at most about 2 hours,at most about 4 hours, at most about 8 hours, at most about 16 hours, atmost about 24 hours, at most about 36 hours, at most about 48 hours, atmost about 3 days, at most about 4 days, at most about 5 days, at mostabout 6 days, at most about 7 days, at most about 8 days, at most about9 days, at most about 10 days, at most about 11 days, at most about 12days, at most about 13 days, at most about 14 days, at most about 15days, at most about 1 month, at most about 2 months, at most about 3months, at most about 4 months, at most about 5 months, at most about 6months, or at most about 1 year after administration of the compositioncomprising the first and second nucleic acid. In some embodiments, thenon-invasive imaging method may be performed multiple times afteradministration of the composition comprising the first and secondnucleic acid (e.g. to monitor synthetic biomarker levels over time). Thenon-invasive imaging method may be performed at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, or 25 times after administration of thecomposition comprising the first and second nucleic acid. Thenon-invasive imaging method may be performed weekly or monthly followingafter administration of the composition comprising the first and secondnucleic acid.

In some cases, the first polypeptide or nucleic acid biomarker and/orthe second polypeptide or nucleic acid biomarker may be detected in abiological sample from the subject. The biological sample may be asample collected by a non-invasive method from the subject. Exemplarynon-invasive samples include, but are not limited to, saliva, sputum,sweat, urine, stool, semen, cervicovaginal secretions, breast milk,rheum, tears, and cheek epithelial swabs. The biological sample may be asample collected by a minimally-invasive method from the subject.Exemplary minimally-invasive samples include but are not limited toblood samples (e.g. obtained by venipuncture or capillary tube), pleuralfluid samples (e.g. obtained by thoracentesis), amniotic fluid samples(e.g. obtained by amniocentesis), and gastric fluid samples (e.g.obtained by gastric lavage). The biological sample may be a sampleobtained by biopsy, such as a skin biopsy sample (e.g. obtained bypunch, shave, saucerization, wedge, incisional, or excisional biopsy), abone marrow sample (e.g. obtained by aspiration biopsy), a lymph node orbreast biopsy (e.g. obtained by fine-needle aspiration, core needlebiopsy, vacuum assisted biopsy, or image-guided biopsy), a surgicalbiopsy sample (e.g. of an internal organ obtained by excisional orincisional biopsy), or a mouth, GI-tract, lung, bladder, or urinarytract biopsy (e.g. obtained by endoscopy). In some cases, the biologicalsample may be obtained a certain period of time after administration ofthe composition inducing expression of the synthetic biomarker. Thebiological sample may be obtained at least about 15 minutes, at leastabout 30 minutes, at least about 1 hour, at least about 2 hours, atleast about 4 hours, at least about 8 hours, at least about 16 hours, atleast about 24 hours, at least about 36 hours, at least about 48 hours,at least about 3 days, at least about 4 days, at least about 5 days, atleast about 6 days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, at least about 14 days, atleast about 15 days, at least about 1 month, at least about 2 months, atleast about 3 months, at least about 4 months, at least about 5 months,or at least about 6 months after administration of the compositioncomprising the first and second nucleic acid. The biological sample maybe obtained at most about 15 minutes, at most about 30 minutes, at mostabout 1 hour, at most about 2 hours, at most about 4 hours, at mostabout 8 hours, at most about 16 hours, at most about 24 hours, at mostabout 36 hours, at most about 48 hours, at most about 3 days, at mostabout 4 days, at most about 5 days, at most about 6 days, at most about7 days, at most about 8 days, at most about 9 days, at most about 10days, at most about 11 days, at most about 12 days, at most about 13days, at most about 14 days, at most about 15 days, at most about 1month, at most about 2 months, at most about 3 months, at most about 4months, at most about 5 months, or at most about 6 months afteradministration of the composition comprising the first and secondnucleic acid. In some embodiments, the biological sample may beobtained, and any biomarker detection protocols performed multiple timespost after administration of the composition comprising the first andsecond nucleic acid (e.g. to monitor synthetic biomarker levels overtime). The biological sample may be obtained at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, or 25 times after administration of thecomposition comprising the first and second nucleic acid. The biologicalsample may be obtained weekly or monthly following after administrationof the composition comprising the first and second nucleic acid.

In some cases, the method may comprise detecting the first or the secondnucleic acid biomarker by a specific nucleic acid detection method. Thefirst or the second nucleic acid biomarker may be detected bysequencing. Sequencing methods may include: Next Generation sequencing,high-throughput sequencing, pyrosequencing, classic Sanger sequencingmethods, sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (LifeTechnologies/Thermo-Fisher), massively-parallel sequencing, clonalsingle molecule Array (Solexa), shotgun sequencing, Maxim-Gilbertsequencing, and primer walking.

In some cases, the first or the second nucleic acid biomarker may bedetected by “real time amplification” methods also known as quantitativePCR (qPCR) or Taqman (see, e.g., U.S. Pat. No. 5,210,015 to Gelfand,U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No. 5,863,736 toHaaland, as well as Heid, C. A., et al., Genome Research, 6:986-994(1996); Gibson, U. E. M, et al., Genome Research 6:995-1001 (1996);Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991);and Livak, K. J., et al., PCR Methods and Applications 357-362 (1995)).The basis for this method of monitoring the formation of amplificationproduct is to measure continuously PCR product accumulation using adual-labeled fluorogenic oligonucleotide probe. The probe used in suchassays is typically a short (ca. 20-25 bases) polynucleotide that islabeled with two different fluorescent dyes. The 5′ terminus of theprobe is typically attached to a reporter dye and the 3′ terminus isattached to a quenching dye. The probe is designed to have at leastsubstantial sequence complementarity with a site on the target mRNA ornucleic acid derived from. Upstream and downstream PCR primers that bindto flanking regions of the locus are also added to the reaction mixture.When the probe is intact, energy transfer between the two fluorophoresoccurs and the quencher quenches emission from the reporter. During theextension phase of PCR, the probe is cleaved by the 5′ nuclease activityof a nucleic acid polymerase such as Taq polymerase, thereby releasingthe reporter from the polynucleotide-quencher and resulting in anincrease of reporter emission intensity which can be measured by anappropriate detector. The recorded values can then be used to calculatethe increase in normalized reporter emission intensity on a continuousbasis and ultimately quantify the amount of the mRNA being amplified.

In some embodiments, for qPCR or Taqman detection, an RT-PCR step mayfirst be performed to generate cDNA from cellular RNA. Suchamplification by RT-PCR can either be general (e.g. amplification withpartially/fully degenerate oligonucleotide primers) or targeted (e.g.amplification with oligonucleotide primers directed against specificgenes which are to be analyzed at a later step).

In some embodiments, qPCR or Taqman may be used immediately following areverse-transcriptase reaction performed on isolated cellular mRNA; thisvariety serves to quantitate the levels of individual mRNAs during qPCR.

In some embodiments, for qPCR or Taqman detection or RNA sequencing, a“pre-amplification” step may be first performed on cDNA transcribed fromcellular RNA. This serves to increase signal in conditions where thenatural level of the RNA/cDNA to be detected is very low. Suitablemethods for pre-amplification include but are not limited LM-PCR, PCRwith random oligonucleotide primers (e.g. random hexamer PCR), PCR withpoly-A specific primers, and any combination thereof. Thepre-amplification may be either general or targeted in the same way asthe reverse-transcription reaction described above.

RNA levels may also be measured without amplification by hybridizationto a probe, for example, using a branched nucleic acid probe, such as aQuantiGene® Reagent System from Panomics.

Heterodimer-based Synthetic Biomarker Design

In some aspects, the present disclosure provides for a compositioncomprising a first nucleic acid sequence encoding a first polypeptideand a second nucleic acid sequence encoding a second polypeptide,wherein the composition is configured such that when the composition isin a cell: (i) the cell expresses the first nucleic acid sequence toyield the first polypeptide; (ii) the cell expresses the second nucleicacid sequence to yield the second polypeptide; and (iii) the firstpolypeptide and the second polypeptide expressed by the cell areconfigured to combine to form a heterodimer protein. In some cases, thefirst polypeptide and the second polypeptide may be on independentgenetic constructs. In some cases, the first polypeptide and the secondpolypeptide may be on independent genetic constructs.

In some cases, the heterodimer protein may be a derivative of anaturally occurring heterodimer or a natural enzyme or autofluorescentprotein split into two complementing polypeptide halves. Examples ofsuch systems include, but are not limited to, an FRB/FKBP12 heterodimer,a split luciferase protein, or a split GFP protein.

In some cases when the heterodimer protein may be a derivative of anaturally occurring heterodimer (e.g. the FRB/FKBP12 pair) each half ofthe heterodimer protein are linked to complementary halves of an enzymeor detection pair, such that dimerization of the heterodimer activatesthe enzyme or allows detection of the detection pair. In some cases,each half of the heterodimer protein may be linked to a splitrecombinase, such as a Cre recombinase, that may activate expression ofan additional element (e.g. a synthetic biomarker or a therapeuticmolecule) when its activity is reconstituted by dimerization of theheterodimer. In some cases, each half of the heterodimer protein may belinked to one of two autofluorescent proteins forming a FRET pair, suchthat FRET may be detected when the heterodimer is formed.

In some cases, the first nucleic acid sequence and the second nucleicacid sequence may be operably linked to a first genetic element and asecond genetic element, wherein both the first genetic element and thesecond genetic element may be selectively activated to express the firstand the second polypeptide in a same diseased cell type. The first orsecond genetic element may be a promoter, an enhancer, or a miRNAbinding site. Exemplary promoters include, but are not limited to,Survivin promoter (BIRC5), a CXCR4 promoter, an ATP binding cassettesubfamily C member 4 (ABCC4) promoter, an anterior gradient 2, proteindisulphide isomerase family member (AGR2) promoter, activation inducedcytidine deaminase (AICDA) promoter, an UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin3 (CDH3) promoter, a CEA cell adhesion molecule 5 (CEACAM5) promoter, acentromere protein F (CENPF) promoter, a centrosomal protein 55 (CEP55)promoter, a claudin 3 (CLDN3) promoter, a claudin 4 (CLDN4) promoter, acollagen type XI alpha 1 chain (COL11A1) promoter, a collagen type Ialpha 1 chain (COL1A1) promoter, a cystatin SN (CST1) promoter, adenticleless E3 ubiquitin protein ligase homolog (DTL) promoter, afamily with sequence similarity 111 member B (FAM111B) promoter, aforkhead box A1 (FOXA1) promoter, a kinesin family member 20A (KIF20A),a laminin subunit gamma 2 (LAMC2) promoter, a mitotic spindlepositioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group IID (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof. Exemplary miRNAs binding sites include, but arenot limited to, at least one miR-15, miR-16, let-7, miR-122 or miR-34binding sequence.

In some cases, the genetic constructs the first and the secondpolypeptide are encoded on may be a vector. Exemplary vectors includeany of the vectors described herein.

In some aspects, the present disclosure provides for a method ofdetecting or treating a diseased cell, comprising administering acomposition, wherein the composition comprises: a first nucleic acidsequence encoding a first polypeptide and a second nucleic acid sequenceencoding a second polypeptide, wherein the composition is configuredsuch that when the composition is in a cell: (i) the cell expresses thefirst nucleic acid sequence to yield the first polypeptide; (ii) thecell expresses the second nucleic acid sequence to yield the secondpolypeptide; and (iii) the first polypeptide and the second polypeptideexpressed by the cell are configured to combine to form a heterodimerprotein. In some cases, the composition is administered intravenously,subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally,inhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-175). In someembodiments, the composition is administered into at least one of thecervical, epitrochlear, supraclavicular, cervical, axillary,mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteallymph nodes. In some cases, lymph-node based administration may serve asa method of centralized local delivery to a tissue region. In somecases, the method may further comprise detecting the heterodimerprotein.

In some cases, the detecting comprises a non-invasive detection methodperformed on the subject. Exemplary non-invasive detection methods (e.g.for autofluorescent or luminescent protein) include, but are not limitedto, SPECT imaging and bioluminescent imaging. The imaging method may beperformed at least about 15 minutes, at least about 30 minutes, at leastabout 1 hour, at least about 2 hours, at least about 4 hours, at leastabout 8 hours, at least about 16 hours, at least about 24 hours, atleast about 36 hours, at least about 48 hours, at least about 3 days, atleast about 4 days, at least about 5 days, at least about 6 days, atleast about 7 days, at least about 8 days, at least about 9 days, atleast about 10 days, at least about 11 days, at least about 12 days, atleast about 13 days, at least about 14 days, at least about 15 days, atleast about 1 month, at least about 2 months, at least about 3 months,at least about 4 months, at least about 5 months, at least about 6months, or at least about 1 year after administration of the compositionencoding the heterodimer protein. The imaging method may be performed atmost about 15 minutes, at most about 30 minutes, at most about 1 hour,at most about 2 hours, at most about 4 hours, at most about 8 hours, atmost about 16 hours, at most about 24 hours, at most about 36 hours, atmost about 48 hours, at most about 3 days, at most about 4 days, at mostabout 5 days, at most about 6 days, at most about 7 days, at most about8 days, at most about 9 days, at most about 10 days, at most about 11days, at most about 12 days, at most about 13 days, at most about 14days, at most about 15 days, at most about 1 month, at most about 2months, at most about 3 months, at most about 4 months, at most about 5months, at most about 6 months, or at most about 1 year afteradministration of the composition encoding the heterodimer protein. Insome embodiments, the imaging method may be performed multiple timespost after administration of the composition encoding the heterodimerprotein (e.g. to monitor synthetic biomarker levels over time). Theimaging method may be performed at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, or 25 times after administration of the compositionencoding the heterodimer protein. The imaging method may be performedweekly or monthly following after administration of the compositionencoding the heterodimer protein.

In some cases, the detecting may comprise detecting the heterodimerprotein from a biological sample from the subject. The biological samplemay be a sample collected by a non-invasive method from the subject.Exemplary non-invasive samples include, but are not limited to, saliva,sputum, sweat, urine, stool, semen, cervicovaginal secretions, breastmilk, rheum, tears, and cheek epithelial swabs. The biological samplemay be a sample collected by a minimally-invasive method from thesubject. Exemplary minimally-invasive samples include, but are notlimited to, blood samples (e.g. obtained by venipuncture or capillarytube), pleural fluid samples (e.g. obtained by thoracentesis), amnioticfluid samples (e.g. obtained by amniocentesis), and gastric fluidsamples (e.g. obtained by gastric lavage). The biological sample may bea sample obtained by biopsy, such as a skin biopsy sample (e.g. obtainedby punch, shave, saucerization, wedge, incisional, or excisionalbiopsy), a bone marrow sample (e.g. obtained by aspiration biopsy), alymph node or breast biopsy (e.g. obtained by fine-needle aspiration,core needle biopsy, vacuum assisted biopsy, or image-guided biopsy), asurgical biopsy sample (e.g. of an internal organ obtained by excisionalor incisional biopsy), or a mouth, GI-tract, lung, bladder, or urinarytract biopsy (e.g. obtained by endoscopy).

In some cases, the biological sample may be obtained a certain period oftime after administration of the composition inducing expression of thesynthetic biomarker. The biological sample may be obtained at leastabout 15 minutes, at least about 30 minutes, at least about 1 hour, atleast about 2 hours, at least about 4 hours, at least about 8 hours, atleast about 16 hours, at least about 24 hours, at least about 36 hours,at least about 48 hours, at least about 3 days, at least about 4 days,at least about 5 days, at least about 6 days, at least about 7 days, atleast about 8 days, at least about 9 days, at least about 10 days, atleast about 11 days, at least about 12 days, at least about 13 days, atleast about 14 days, at least about 15 days, at least about 1 month, atleast about 2 months, at least about 3 months, at least about 4 months,at least about 5 months, or at least about 6 months after administrationof the composition encoding the heterodimer protein. The biologicalsample may be obtained at most about 15 minutes, at most about 30minutes, at most about 1 hour, at most about 2 hours, at most about 4hours, at most about 8 hours, at most about 16 hours, at most about 24hours, at most about 36 hours, at most about 48 hours, at most about 3days, at most about 4 days, at most about 5 days, at most about 6 days,at most about 7 days, at most about 8 days, at most about 9 days, atmost about 10 days, at most about 11 days, at most about 12 days, atmost about 13 days, at most about 14 days, at most about 15 days, atmost about 1 month, at most about 2 months, at most about 3 months, atmost about 4 months, at most about 5 months, or at most about 6 monthsafter administration of the composition encoding the heterodimerprotein. In some embodiments, the biological sample may be obtained, andany biomarker detection protocols performed multiple times afteradministration of the composition encoding the heterodimer protein. Thebiological sample may be obtained at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, or 25 times after administration of the compositionencoding the heterodimer protein. The biological sample may be obtainedweekly or monthly following after administration of the compositionencoding the heterodimer protein. The heterodimer protein in thebiological sample may be detected by fluorescence assay, FRET assay,TR-FRET assay, or luminescent assay.

Alternatively or additionally, the heterodimer protein may be detectedin a heterodimer-specific immunodetection assay. Several methods anddevices are well known for determining levels of proteins includingimmunoassays such as described in e.g., U.S. Pat. Nos. 6,143,576;6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615;5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792.These assays include various sandwich, competitive, or non-competitiveassay formats, to generate a signal that is related to the presence oramount of a protein analyte of interest. Any suitable immunoassay may beutilized, for example, lateral flow, enzyme-linked immunoassays (ELISA),radioimmunoassays (RIAs), competitive binding assays, or any combinationthereof.

Ex-Vivo Constructs and Methods for Synthetic Biomarkers to Use inDisease Detection, Monitoring, or Diagnosis

In some aspects, the present disclosure provides for a compositioncomprising a non-naturally occurring recombinant genetic constructcomprising a sequence encoding a polypeptide or nucleic acid sequence,wherein the sequence comprises a first promoter that selectively drivesexpression of the polypeptide or nucleic acid biomarker sequence in aplurality of different types of diseased cells isolated from a subjectwhen transduced into the cells ex vivo.

In some cases, the composition may comprise the cells transduced withthe recombinant genetic construct. In some cases, the plurality ofdifferent types of cells are diseased or disordered cells. In somecases, the cells are blood cells, lymphocytes, leukocytes, epithelialcells, gastrointestinal cells, placental cells, amniotic cells, lungepithelial cells, urinary epithelial cells, or kidney cells.

In some cases, the diseased or disordered cells may be cancerous cell,cells indicative of an autoimmune disease (e.g. T-cells or lymphocyteswith self-directed activity, or normal cells damaged by autoimmunity),TACs, or cells indicative of a neurodegenerative disease (e.g. cellsbearing a toxic amyloid or proximal to a toxic amyloid). Exemplarycancers, autoimmune, and neurodegenerative diseases include any of thediseases described herein. In some cases, the diseased or disorderedcells may be virally-infected cells. Exemplary viral infections include,but are not limited to, those caused by HIV, hepatitis C virus,hepatitis B virus, hepatitis D virus, herpesviruses, Epstein-Barr virus,cytomegalovirus, and human T-lymphotropic virus type III.

In some cases, the first promoter may be a promoter activated in thecells when the cells are in a diseased state. The first promoter may bea pan-tumor specific promoter. In some cases, the first promoter is acancer-specific promoter. In some cases, the first promoter is aSurvivin promoter (BIRC5), a CXCR4 promoter, an ATP binding cassettesubfamily C member 4 (ABCC4) promoter, an anterior gradient 2, proteindisulphide isomerase family member (AGR2) promoter, activation inducedcytidine deaminase (AICDA) promoter, an UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a cadherin3 (CDH3) promoter, a CEA cell adhesion molecule 5 (CEACAM5) promoter, acentromere protein F (CENPF) promoter, a centrosomal protein 55 (CEP55)promoter, a claudin 3 (CLDN3) promoter, a claudin 4 (CLDN4) promoter, acollagen type XI alpha 1 chain (COL11A1) promoter, a collagen type Ialpha 1 chain (COL1A1) promoter, a cystatin SN (CST1) promoter, adenticleless E3 ubiquitin protein ligase homolog (DTL) promoter, afamily with sequence similarity 111 member B (FAM111B) promoter, aforkhead box A1 (FOXA1) promoter, a kinesin family member 20A (KIF20A),a laminin subunit gamma 2 (LAMC2) promoter, a mitotic spindlepositioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group BD (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof.

In some cases, the recombinant genetic construct for detection ex vivomay comprise retroviral, lentiviral, or adenoviral packaging elements orlong terminal repeats. The recombinant genetic construct may be a CELiDvector. The recombinant genetic construct may be a vector derived from abacteriophage or plant, invertebrate, or animal (including human) virussuch as an adeno-associated viral vector (e.g. AAV1, AAV2, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, or pseudotyped combination thereof such asAAV2/5, AAV2/2, AAV-DJ, or AAV-DJ8), a retroviral vector (e.g. MLV orself-inactivating or SIN versions thereof, or pseudotyped versionsthereof), herpesvirus (e.g. HSV- or EBV-based), a lentivirus vector(e.g. HIV-, FIV-, or EIAV-based, or pseudotyped versions thereof), or anadenoviral vector (e.g. Ad5-based, including replication-deficient,replication-competent, or helper-dependent versions thereof). Therecombinant genetic construct may also be a packaging vector compatiblewith any of these viral systems.

The vector may be a non-viral vector. The non-viral vector may be aminicircle vector. The minicircle may be a self-replicating minicircle.The self-replicating minicircle may comprise an S/MAR element. Thenon-viral vector may be a nanoplasmid or mini-intronic plasmid (MIP).MIP places the bacterial replication origin and any selectable marker asan intron in the transgene expression cassette. Further, MIP may keepthe juxtaposition of the 5′ and 3′; ends of transgene expressioncassette as in minicircle (see e.g. Lu et al., a mini-intronic plasmid(MIP): a novel robust transgene expression vector in vivo and in vitro,mol. Ther. 2013 May; 21(5): 954-963)

In some cases, the polypeptide or nucleic acid sequence selectivelyexpressed when transduced into ex vivo may be selected from the groupconsisting of a photoacoustic reporter, a bioluminescent reporter, anautofluorescent reporter, a chemiluminescent reporter, a luminescentreporter, a colorimetric reporter, a quantifiable nucleic acid, and anycombination thereof. Autofluorescent reporters include GFP, mCherry, orderivatives thereof. Colorimetric reporters include pigment-producingenzymes such as β-galactosidase (e.g. in combination with administrationof X-gal), and tyrosinase. Bioluminescent, chemiluminescent orluminescent reporters include luciferases (e.g. in combination withadministration of coelenterazines described herein), including Gaussialuciferases, Renilla luciferases, and Photinus luciferases (e.g.including the engineered Ppy RE8 and RE9 versions described in Branchiniet al. Anal. Biochem. 396(2010): 290-297). Reporters detectable byphotoacoustic imaging include the pigment-producing enzymes such asβ-galactosidase (e.g. in combination with administration of X-gal) andtyrosinase, autofluorescent proteins (e.g. GFP, mCherry, or derivativesthereof), non-fluorescent GFP-like chromoproteins (e.g. aeCP597 andcjBlue and derivatives thereof), bacteriophytochrome-based near-infraredfluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713,iRFP720, iRFP713/V256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry,GAF-FP, BphP1-FP/C20S, or AphB variants), and reversiblyphoto-switchable proteins (e.g. Dronpa, Dronpa-M159T, and BphP1 orvariants thereof). The quantifiable nucleic acid may be a ribozyme, aself-splicing intron, an RNA hairpin, a microRNA, or barcoded versionsthereof, or other types of quantifiable RNA. The quantifiable nucleicacid may comprise a unique sequence detectable by quantitative PCR orhybridization-based techniques. When the polypeptide or nucleic acid isa polypeptide, the polypeptide may comprise an N-terminal secretionsignal sequence (e.g. the N-terminal signal peptide from CD33 or CD8a).

In some cases, the composition may have a certain diagnostic efficiency,wherein the diagnostic efficiency is measured in a diseased cellpreferentially over expression of said biomarker in non-diseased cellsin said subject such that a relative ratio of said biomarker expressedin said diseased cell over said non-diseased cells is greater than 1.0;(b) detecting said biomarker; and (c) using said biomarker detected in(b) to determine that said subject has said diseased cell at an accuracyof at least 90%.

In some cases, the composition administered to the cells ex vivo maycomprise a second polypeptide or nucleic acid that modulates theproliferation of diseased or disordered cells. The second polypeptidemay be under the control of a second promoter that selectively drivesexpression of the second polypeptide or nucleic acid in the diseased ordisordered cell. The second promoter may be a pan-cancer specificpromoter. The second promoter may be a cancer-specific promoter. Thepromoter may be any of the specific promoters described herein. Thesecond polypeptide may comprise a transforming agent or a growth factor.The transforming agent may comprise telomerase or SV40 large T antigen.The growth factor may be e.g. EGF, PDGF, FGF, HGH, or IGF-1.

In some aspects, the present disclosure provides for a method fordetecting a diseased or disordered cell ex-vivo, comprising deliveringex vivo a non-naturally occurring recombinant genetic construct to apopulation of cells isolated from a subject, wherein the non-naturallyoccurring recombinant genetic construct comprises: a sequence encoding apolypeptide or nucleic acid sequence, wherein the sequence comprises afirst promoter that selectively drives expression of the polypeptide ornucleic acid sequence in a plurality of different types of cellsisolated from a subject when transduced into the cells.

In some aspects, the present disclosure provides for a method fordetecting a subject's disease or absence thereof, comprising contactingone or more cells of said subject with a genetic construct ex-vivo,wherein said genetic construct comprises a disease-activated promoteroperably linked to a barcode molecule and said disease-activatedpromoter drives expression of said barcode molecule in a cell affectedby said disease; quantifying an expression level of said barcodemolecule; and detecting said disease or absence thereof based on saidexpression level.

In some cases, the method for detecting a diseased or disordered cellex-vivo may be capable of detecting a particular number of diseasedcells in a background of normal cells. In some embodiments, the methodmay be capable of detecting about at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 20, at least about 30,at least about 40, at least about 50, at least about 60, at least about70, at least about 80, at least about 90, at least about 100, at leastabout 200, at least about 300, at least about 400, or at least about 500diseased cells per 5 million normal cells. In some embodiments, thenormal cells are blood cells (e.g. PBMCs).

In some cases, the method may comprise isolating a biological samplecomprising the cells from the subject. The biological sample may be asample collected by a non-invasive method from the subject. Exemplarynon-invasive samples include, but are not limited to, samples comprisedof naturally shed bodily substances or non-destructive scraping ofexternally accessible tissues, such as saliva, sputum, sweat, urine,stool, semen, mucus, cervicovaginal secretions, breast milk, rheum,tears, and cheek epithelial swabs. The biological sample may be a samplecollected by a minimally-invasive method from the subject. Exemplaryminimally-invasive samples include, but are not limited to, bloodsamples or fractions thereof (e.g. obtained by venipuncture or capillarytube), pleural fluid samples (e.g. obtained by thoracentesis), amnioticfluid samples (e.g. obtained by amniocentesis), and gastric fluidsamples (e.g. obtained by gastric lavage). The biological sample may bea sample obtained by biopsy, such as a skin biopsy sample (e.g. obtainedby punch, shave, saucerization, wedge, incisional, or excisionalbiopsy), a bone marrow sample (e.g. obtained by aspiration biopsy), alymph node or breast biopsy (e.g. obtained by fine-needle aspiration,core needle biopsy, vacuum assisted biopsy, or image-guided biopsy), asurgical biopsy sample (e.g. of an internal organ obtained by excisionalor incisional biopsy), or a mouth, GI-tract, lung, bladder, or urinarytract biopsy (e.g. obtained by endoscopy).

In some cases, the method may comprise culturing the population of cellsfor a certain period of time after the recombinant genetic construct isdelivered to the cells. The population of cells may be cultured forleast about 15 minutes, at least about 30 minutes, at least about 1hour, at least about 2 hours, at least about 4 hours, at least about 8hours, at least about 16 hours, at least about 24 hours, at least about36 hours, at least about 48 hours, at least about 3 days, at least about4 days, at least about 5 days, at least about 6 days, at least about 7days, at least about 8 days, at least about 9 days, at least about 10days, at least about 11 days, at least about 12 days, at least about 13days, at least about 14 days, at least about 15 days, or at least about1 month after delivery of the genetic construct to the cells. Thepopulation of cells may be cultured for most about 15 minutes, at mostabout 30 minutes, at most about 1 hour, at most about 2 hours, at mostabout 4 hours, at most about 8 hours, at most about 16 hours, at mostabout 24 hours, at most about 36 hours, at most about 48 hours, at mostabout 3 days, at most about 4 days, at most about 5 days, at most about6 days, at most about 7 days, at most about 8 days, at most about 9days, at most about 10 days, at most about 11 days, at most about 12days, at most about 13 days, at most about 14 days, at most about 15days, or at most about 1 month after delivery of the genetic constructto the cells.

In some cases, the method may comprise detecting the polypeptide ornucleic acid sequence. The detecting may occur before or after culturingthe population of cells. The detecting may comprise a photoacoustic, abioluminescent, fluorescent reporter, chemiluminescent, luminescent,colorimetric, or nucleic acid assay. The detecting may also comprise animmunoassay. Immunoassays include those described in e.g., U.S. Pat.Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792. Immunoassays include various sandwich, competitive, ornon-competitive assay formats, which generate a signal that is relatedto the presence or amount of a protein analyte of interest. Any suitableimmunoassay may be utilized, for example, lateral flow, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

The method of detection may comprise sequencing. Sequencing methods mayinclude: Next Generation sequencing, high-throughput sequencing,pyrosequencing, classic Sanger sequencing methods,sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (LifeTechnologies/Thermo-Fisher), massively-parallel sequencing, clonalsingle molecule Array (Solexa), shotgun sequencing, Maxim-Gilbertsequencing, and primer walking.

The detection may comprise a “real time amplification” method also knownas quantitative PCR (qPCR) or Taqman (see, e.g., U.S. Pat. No. 5,210,015to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No.5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362(1995)). The basis for this method of monitoring the formation ofamplification product is to measure continuously PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe. Theprobe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye. The probe is designed tohave at least substantial sequence complementarity with a site on thetarget mRNA or nucleic acid derived from. Upstream and downstream PCRprimers that bind to flanking regions of the locus are also added to thereaction mixture. When the probe is intact, energy transfer between thetwo fluorophores occurs and the quencher quenches emission from thereporter. During the extension phase of PCR, the probe is cleaved by the5′ nuclease activity of a nucleic acid polymerase such as Taqpolymerase, thereby releasing the reporter from thepolynucleotide-quencher and resulting in an increase of reporteremission intensity which can be measured by an appropriate detector. Therecorded values can then be used to calculate the increase in normalizedreporter emission intensity on a continuous basis and ultimatelyquantify the amount of the mRNA being amplified.

In some embodiments, for qPCR or Taqman detection, an RT-PCR step mayfirst be performed to generate cDNA from cellular RNA. Suchamplification by RT-PCR can either be general (e.g. amplification withpartially/fully degenerate oligonucleotide primers) or targeted (e.g.amplification with oligonucleotide primers directed against specificgenes which are to be analyzed at a later step).

In some embodiments, qPCR or Taqman may be used immediately following areverse-transcriptase reaction performed on isolated cellular mRNA; thisvariety serves to quantitate the levels of individual mRNAs during qPCR.

In some embodiments, for qPCR or Taqman detection or RNA sequencing, a“pre-amplification” step may be first performed on cDNA transcribed fromcellular RNA. This serves to increase signal in conditions where thenatural level of the RNA/cDNA to be detected is very low. Suitablemethods for pre-amplification include but are not limited LM-PCR, PCRwith random oligonucleotide primers (e.g. random hexamer PCR), PCR withpoly-A specific primers, and any combination thereof. Thepre-amplification may be either general or targeted in the same way asthe reverse-transcription reaction described above.

Improved Biomarkers, Construct Design, and Methods for Disease StageIndication

In some aspects, the present disclosure provides for a compositioncomprising a vector, wherein the vector comprises a plurality ofdifferent promoters operably linked to a plurality of different nucleicacid sequences, wherein each the promoter drives expression of theplurality of nucleic acid sequences in a cell to yield a plurality ofpolypeptides or nucleic acid biomarker sequences, wherein levels ofindividual polypeptides or nucleic acid biomarkers of the plurality ofnucleic acid sequences are indicative of a stage of a disease of thecell. In some cases, the stage of the disease of the cell is diseased,non-diseased or an intermediate state. In some cases, the plurality ofdifferent promoters may be included on a plurality of independentgenetic constructs or vectors that are administered simultaneously orseparately. In some embodiments, the plurality of independent geneticconstructs administered separately are administered within 8, 16, 24,36, 48, 60, or 72 hours of one another. In some embodiments, diseasestage may be assessed by dissemination of cancer cells away from theirtissue of origin via metastasis to distal tissues. In cases such asthis, the plurality of different promoters may comprise at least apromoter with high cancer specificity in the initial tissue site (e.g.breast, when breast cancer is being staged) and a promoter active withhigh specificity at a common metastatic site (e.g. lung, spleen, liver)different from the initial site. In some cases, the plurality ofdifferent promoters may comprise at least a promoter with high cancerspecificity in the initial tissue site (e.g. breast, when breast canceris being staged) and multiple promoters active with high specificity atmultiple distinct metastatic sites (e.g. lung, spleen, liver). Thus,such systems may provide activation of more distinct promoters (whichcan be read out by their operably linked biomarkers downstream) as thecancer metastasizes from its home site to metastatic sites, providing anassessment of how widely the tumor has metastasized. In someembodiments, one of the promoter active with high specificity at acommon metastatic site is MMP-2, which provides high expression at allstages at lung cancer but is not overexpressed in breast cancer.

In some cases, the disease may be cancer, an autoimmune disease (e.g. aT-cell or lymphocyte with self-directed activity, or a normal celldamaged by autoimmunity), or a neurodegenerative disease (e.g. a cellbearing a toxic amyloid or proximal to a toxic amyloid). Exemplarycancers include, but are not limited to, carcinomas, sarcomas,lymphomas, leukemias, and adenomas. Carcinomas may arise from cells thatcover internal and external parts of the body such as the lung, breast,and colon. Sarcomas may arise from cells that are located in bone,cartilage, fat, connective tissue, muscle, and other supportive tissues.Lymphomas may arise in the lymph nodes and immune system tissues.Leukemias may arise in the bone marrow and accumulate in thebloodstream. Adenomas may arise in the thyroid, the pituitary gland, theadrenal gland, and other glandular tissues. Specific exemplary examplesof cancer types include suitable for detection with the methodsaccording to the disclosure include acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basalcell carcinoma, bile duct cancer, bladder cancer, bone cancers, braintumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignantglioma, ependymoma, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, breastcancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknownprimary origin, central nervous system lymphoma, cerebellar astrocytoma,cervical cancer, childhood cancers, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic myeloproliferative disorders,colon cancer, cutaneous T-cell lymphoma, desmoplastic small round celltumor, endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma, germ cell tumors, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor,gliomas, hairy cell leukemia, head and neck cancer, heart cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer,intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidneycancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, livercancer, lung cancers, such as non-small cell and small cell lung cancer,lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytomaof bone/osteosarcoma, medulloblastoma, melanomas, mesothelioma,metastatic squamous neck cancer with occult primary, mouth cancer,multiple endocrine neoplasia syndrome, myelodysplastic syndromes,myeloid leukemia, nasal cavity and paranasal sinus cancer,nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-smallcell lung cancer, oral cancer, oropharyngeal cancer,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer,ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer,pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonaryblastoma, plasma cell neoplasia, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renalpelvis and ureter transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skincarcinoma merkel cell, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer,thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor(gestational), cancers of unknown primary site, urethral cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia,and Wilms tumor.

In some cases, the disease may be a viral infection cell. Exemplaryviral infections include, but are not limited to, those caused by HIV,hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesviruses,Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus typeIII.

In some cases, when the disease is cancer, the plurality of differentpromoters may comprise a first promoter activated in an early stage ofcancer. In some cases, the plurality of different promoters may comprisea second promoter activated in an intermediate stage of cancer. In somecases, the plurality of different promoters comprises a third promoteractivated in a late stage of cancer.

In some cases, the disease may be an autoimmune disease. Exemplaryautoimmune diseases include, but are not limited to, Achalasia,Addison's disease, Adult Still's disease, Agammaglobulinemia, Alopeciaareata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmunedysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis,Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmuneoophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmuneretinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN),Baló disease, Behcet's disease, Benign mucosal pemphigoid, Bullouspemphigoid, Castleman disease (CD), Celiac disease, Chagas disease,Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronicrecurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS)or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan'ssyndrome, Cold agglutinin disease, Congenital heart block, Coxsackiemyocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis,Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus,Dressler's syndrome, Endometriosis, Eosinophilic esophagitis (EoE),Eosinophilic fasciitis, Erythema nodosum, Essential mixedcryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis,Giant cell arteritis (temporal arteritis), Giant cell myocarditis,Glomerulonephritis, Goodpasture's syndrome, Granulomatosis withPolyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto'sthyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpesgestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa(HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy,IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP),Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenilearthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM),Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis,Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgAdisease (LAD), Lupus, Lyme disease chronic, Meniere's disease,Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy(MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis,Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocularcicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR),PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis(peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheralneuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMSsyndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III,Polymyalgia rheumatica, Polymyositis, Postmyocardial infarctionsyndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis,Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis,Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum,Raynaud's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy,Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitonealfibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidtsyndrome, Scleritis, Scleroderma, Sjögren's syndrome, Sperm & testicularautoimmunity, Stiff person syndrome (SPS), Subacute bacterialendocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transversemyelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiatedconnective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, andVogt-Koyanagi-Harada Disease.

In some cases, the disease may a neurodegenerative disease.Neurodegenerative diseases include, but are not limited to, Multiplesclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), andAmyotrophic lateral sclerosis (ALS), or neurodegeneration due toinfection by viruses of families Herpesviridae, Polyomaviridae,Bornaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae,Flaviviridae, Picornaviridae, or Retroviridae (see Zhou et al. Virol J.2013; 10: 172).

In some cases, the nucleic acid biomarker may be e.g. a natural orengineered miRNA, an RNA hairpin, RNA aptamers or barcoded versionsthereof.

In some cases, the vector provided in the composition to detect thestage of disease may be any of the vectors described herein.

In some cases, at least one of the plurality of polypeptides maycomprise a polypeptide detectable by non-invasive imaging. Suchnon-invasive imagine methods include MRI imaging, PET imaging, SPECTimaging, photoacoustic imaging, and bioluminescent imaging. Syntheticbiomarkers detectable by MRI imaging include, but are not limited to,polypeptide contrast agents, such as ferritin (or mutants thereof, suchas Pyrococcus furiousus ferritin mutants L55P, F57S, or F123S), orlanthanide-binding proteins (or engineered fusions thereof, such as theLBT-ubiquitin fusions described in Daughtry et al. ChemBioChem 2012, 13,2567-2574). Synthetic biomarkers detectable by PET or SPECT imaginginclude the human sodium iodide symporter (e.g. in conjunction withadministration of PET-active iodine/iodide isotopes, see e.g. Penheiteret al. Curr Gene Ther. 2012 February; 12(1): 33-47), HSV-tk or mutantsthereof such as HSV-sr39tk (e.g. in conjunction with administration ofpositron-labeled acycloguanosine or pyrimidine analog PET reporters suchas [18F]FHBG, see Yaghoubi S S et al. Nat Protoc. 2006; 1(6):3069-75),and the dopamine D2 receptor or mutants thereof such as D2R80A orD2R194A (e.g. in conjunction with administration of positron-labeled D2binders such as 3-(2′[18F]-fluoroethyl)-spiperone). Synthetic biomarkersdetectable by photoacoustic imaging include the pigment-producingenzymes such as β-galactosidase (e.g. in combination with administrationof X-gal) and tyrosinase, autofluorescent proteins (e.g. GFP, mCherry,or derivatives thereof), non-fluorescent GFP-like chromoproteins (e.g.aeCP597 and cjBlue and derivatives thereof), bacteriophytochrome-basednear-infrared fluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev,IFP2.0, iRFP713, iRFP720, iRFP713/V256C, iRFP682, iRFP702, iRFP670,mIFP, iBlueberry, GAF-FP, BphP1-FP/C20S, or AphB variants), andreversibly photoswitchable proteins (e.g. Dronpa, Dronpa-M159T, andBphP1 or variants thereof). Synthetic biomarkers detectable bybioluminescent imaging include luciferases (e.g. in combination withadministration of coelenterazines described herein), including Gaussialuciferases, Renilla luciferases, and Photinus luciferases (e.g.including the engineered Ppy RE8 and RE9 versions described in Branchiniet al. Anal. Biochem. 396(2010): 290-297). In some embodiments, thesynthetic biomarker may be a contrast agent, an enzyme producing adetectable molecule, or a transporter driving accumulation of adetectable molecule. The synthetic biomarker may be measured in situwithin subject's body.

In some cases, the barcode molecules may be polypeptides or nucleicacids detectable in a biological sample from the subject. When thebarcode molecule is a polypeptide, the polypeptide may comprise anN-terminal secretion signal sequence (e.g. the N-terminal signal peptidefrom CD33 or CD8a). Exemplary polypeptide biomarkers include, but arenot limited to, photoacoustic reporters, bioluminescent reporters,autofluorescent reporters, chemiluminescent reporters, luminescentreporters, colorimetric reporters, and any combination thereofAutofluorescent reporters include GFP, mCherry, or derivatives thereof.Colorimetric reporters include pigment-producing enzymes such asβ-galactosidase (e.g. in combination with administration of X-gal), andtyrosinase. Bioluminescent, chemiluminescent or luminescent reportersinclude luciferases (e.g. in combination with administration ofcoelenterazines described herein), including Gaussia luciferases,Renilla luciferases, and Photinus luciferases (e.g. including theengineered Ppy RE8 and RE9 versions described in Branchini et al. Anal.Biochem. 396(2010): 290-297). Reporters detectable by photoacousticimaging include the pigment-producing enzymes such as β-galactosidase(e.g. in combination with administration of X-gal) and tyrosinase,autofluorescent proteins (e.g. GFP, mCherry, or derivatives thereof),non-fluorescent GFP-like chromoproteins (e.g. aeCP597 and cjBlue andderivatives thereof), bacteriophytochrome-based near-infraredfluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713,iRFP720, iRFP713/V256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry,GAF-FP, BphP1-FP/C20S, or AphB variants), and reversiblyphoto-switchable proteins (e.g. Dronpa, Dronpa-M159T, and BphP1 orvariants thereof). When the barcode molecule is a nucleic acid sequence,the detectable nucleic acid sequence may comprise, but not be limitedto, a ribozyme, a self-splicing intron, an RNA hairpin, a microRNA, orbarcoded versions thereof, or other types of quantifiable RNA. Thequantifiable nucleic acid sequence may comprise a unique sequencedetectable by quantitative PCR or hybridization-based techniques.

By ascribing an exclusive label to a unique member within a largergroup, barcodes afford the opportunity to identify and quantify thatmember (e.g. expression of a reporter under the control of a particularcancer specific promoter) within the context of a larger and morecomplex mixture of many members (e.g. multiple promoter-reporterconstructs expressed within the same cell), as well as offering theopportunity to isolate a single member from the complex mixture. Forinstance, in the case of barcodes based on nucleic acids, hybridizationof barcodes based on base pairing complementarity may be used to captureand isolate or otherwise reduce the complexity of a mixture by saidcapture event. For barcodes based on peptides, unique features includingimmunocapture or interactions of ligands and receptors may be used tocapture and isolate or otherwise reduce the complexity of a mixture bysaid capture event.

In some aspects, the present disclosure provides for a method fordetecting a stage of disease, comprising administering to a subject acomposition comprising a vector, wherein the vector comprises: aplurality of different promoters operably linked to a plurality ofdifferent nucleic acid sequences, wherein each the promoter drivesexpression of the plurality of nucleic acid sequences in a cell to yielda plurality of polypeptides or synthetic nucleic acid sequences, whereinlevels of individual polypeptides of the plurality of nucleic acidsequences are indicative of a stage of a disease of the cell. In somecases, the stage of the disease of the cell may be diseased,non-diseased or an intermediate state. In some aspects, the presentdisclosure provides for a method for detecting different types ofcancers, comprising administering to a subject a composition comprisinga vector, wherein the vector comprises, a plurality to differentpromoters operably linked to a plurality of different nucleic acidsequences in a cell to yield a plurality of polypeptides or syntheticnucleic acid sequences, wherein levels of individual polypeptides of theplurality of nucleic acid sequences are indicative of a different typeof cancer within the body. In some cases, the cancer detected within thebody may be derived, but not limited to, tissues of the breast, liver,colon, brain, lung, kidney, pancreas, testis, ovaries, blood orcomponents of the blood, bone, stomach, eye, endocrine or neuroendocrinetissues, head and neck, gastrointestinal, musculoskeletal, skin,respiratory, neurologic, or genitourinary, or cancers derived from otherparts of the body.

In some cases, the composition is administered intravenously,subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally,inhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-175). In someembodiments, the composition is administered into at least one of thecervical, epitrochlear, supraclavicular, cervical, axillary,mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteallymph nodes. In some cases, lymph-node based administration may serve asa method of centralized local delivery to a tissue region.

In some cases, when the disease is cancer, the plurality of differentpromoters may comprise a first promoter activated in an early stage ofcancer. In some cases, the plurality of different promoters may comprisea second promoter activated in an intermediate stage of cancer. In somecases, the plurality of different promoters comprises a third promoteractivated in a late stage of cancer. In some cases, the method mayidentify masses of tissue or lesions in the subject as pre-cancerous,benign, dysplastic, or metastatic in nature.

In some cases, the method may comprise isolating a biological samplefrom the subject. The biological sample may be a sample collected by anon-invasive method from the subject. Exemplary non-invasive samplesinclude, but are not limited to, samples comprised of naturally shedbodily substances or non-destructive scraping of externally accessibletissues, such as saliva, sputum, sweat, urine, stool, semen, mucus,cervicovaginal secretions, breast milk, rheum, tears, and cheekepithelial swabs. The biological sample may be a sample collected by aminimally-invasive method from the subject. Exemplary minimally-invasivesamples include, but are not limited to, blood samples or fractionsthereof (e.g. obtained by venipuncture or capillary tube), pleural fluidsamples (e.g. obtained by thoracentesis), amniotic fluid samples (e.g.obtained by amniocentesis), and gastric fluid samples (e.g. obtained bygastric lavage). The biological sample may be a sample obtained bybiopsy, such as a skin biopsy sample (e.g. obtained by punch, shave,saucerization, wedge, incisional, or excisional biopsy), a bone marrowsample (e.g. obtained by aspiration biopsy), a lymph node or breastbiopsy (e.g. obtained by fine-needle aspiration, core needle biopsy,vacuum assisted biopsy, or image-guided biopsy), a surgical biopsysample (e.g. of an internal organ obtained by excisional or incisionalbiopsy), or a mouth, GI-tract, lung, bladder, or urinary tract biopsy(e.g. obtained by endoscopy). In some cases, the biological sample maybe collected period of time after the composition is administered to thesubject.

The population of cells may be cultured for least about 15 minutes, atleast about 30 minutes, at least about 1 hour, at least about 2 hours,at least about 4 hours, at least about 8 hours, at least about 16 hours,at least about 24 hours, at least about 36 hours, at least about 48hours, at least about 3 days, at least about 4 days, at least about 5days, at least about 6 days, at least about 7 days, at least about 8days, at least about 9 days, at least about 10 days, at least about 11days, at least about 12 days, at least about 13 days, at least about 14days, at least about 15 days, or at least about 1 month after deliveryof the genetic construct to the cells. The population of cells may becultured for most about 15 minutes, at most about 30 minutes, at mostabout 1 hour, at most about 2 hours, at most about 4 hours, at mostabout 8 hours, at most about 16 hours, at most about 24 hours, at mostabout 36 hours, at most about 48 hours, at most about 3 days, at mostabout 4 days, at most about 5 days, at most about 6 days, at most about7 days, at most about 8 days, at most about 9 days, at most about 10days, at most about 11 days, at most about 12 days, at most about 13days, at most about 14 days, at most about 15 days, or at most about 1month after delivery of the genetic construct to the cells.

In some cases, the method may comprise detecting the polypeptide ornucleic acid sequence. The detecting may occur before or after culturingthe population of cells. The detecting may comprise a photoacoustic, abioluminescent, fluorescent reporter, chemiluminescent, luminescent,colorimetric, or nucleic acid assay. The detecting may also comprise animmunoassay. Immunoassays include those described in e.g., U.S. Pat.Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792. Immunoassays include various sandwich, competitive, ornon-competitive assay formats, which generate a signal that is relatedto the presence or amount of a protein analyte of interest. Any suitableimmunoassay may be utilized, for example, lateral flow, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

The method of detection may comprise sequencing. Sequencing methods mayinclude: Next Generation sequencing, high-throughput sequencing,pyrosequencing, classic Sanger sequencing methods,sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (LifeTechnologies/Thermo-Fisher), massively-parallel sequencing, clonalsingle molecule Array (Solexa), shotgun sequencing, Maxim-Gilbertsequencing, and primer walking.

The detection may comprise a “real time amplification” method also knownas quantitative PCR (qPCR) or Taqman (see, e.g., U.S. Pat. No. 5,210,015to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No.5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362(1995)). The basis for this method of monitoring the formation ofamplification product is to measure continuously PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe. Theprobe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye. The probe is designed tohave at least substantial sequence complementarity with a site on thetarget mRNA or nucleic acid derived from. Upstream and downstream PCRprimers that bind to flanking regions of the locus are also added to thereaction mixture. When the probe is intact, energy transfer between thetwo fluorophores occurs and the quencher quenches emission from thereporter. During the extension phase of PCR, the probe is cleaved by the5′ nuclease activity of a nucleic acid polymerase such as Taqpolymerase, thereby releasing the reporter from thepolynucleotide-quencher and resulting in an increase of reporteremission intensity which can be measured by an appropriate detector. Therecorded values can then be used to calculate the increase in normalizedreporter emission intensity on a continuous basis and ultimatelyquantify the amount of the mRNA being amplified.

In some embodiments, for qPCR or Taqman detection, an RT-PCR step mayfirst be performed to generate cDNA from cellular RNA. Suchamplification by RT-PCR can either be general (e.g. amplification withpartially/fully degenerate oligonucleotide primers) or targeted (e.g.amplification with oligonucleotide primers directed against specificgenes which are to be analyzed at a later step).

In some embodiments, qPCR or Taqman may be used immediately following areverse-transcriptase reaction performed on isolated cellular mRNA; thisvariety serves to quantitate the levels of individual mRNAs during qPCR.

In some embodiments, for qPCR or Taqman detection or RNA sequencing, a“pre-amplification” step may first be performed on cDNA transcribed fromcellular RNA. This serves to increase signal in conditions where thenatural level of the RNA/cDNA to be detected is very low. Suitablemethods for pre-amplification include but are not limited LM-PCR, PCRwith random oligonucleotide primers (e.g. random hexamer PCR), PCR withpoly-A specific primers, and any combination thereof. Thepre-amplification may be either general or targeted in the same way asthe reverse-transcription reaction described above.

Improved Synthetic Biomarker Design and Method for Expression LeakinessReduction

In some aspects, the present disclosure provides for a compositioncomprising an engineered nucleic acid encoding an expressible reportergene that exhibits about 10% or less expression in normal cells versusdiseased cells when compared to a recombinant nucleic acid comprising areporter gene comprising a nucleic acid sequence of SEQ ID NO: 1 or SEQID NO: 2.

In some cases, the engineered nucleic acid may comprise a pan-tumorspecific promoter operably linked to the expressible reporter gene. Insome cases, the pan-tumor specific promoter may comprise atranscriptional response element. The transcriptional response elementmay comprise a modified p53 response element. A modification within themodified p53 response element may result in decreased promoter activityin normal cells relative to diseased cells. A modification within themodified p53 response element may result in increased promoter activityin diseased cells relative to normal cells.

In some cases, the engineered nucleic acid encoding the expressiblereporter gene may be any of the vectors described herein.

In some cases, the reporter gene may encode a detectable polypeptide ora detectable nucleic acid. The detectable nucleic acid biomarker may bea ribozyme, a self-splicing intron, an RNA hairpin, a microRNA, RNAaptamers or barcoded versions thereof, or other types of quantifiableRNA. The quantifiable nucleic acid may comprise a unique sequencedetectable by quantitative PCR or hybridization-based techniques.

The reporter gene may encode a photoacoustic reporter, a bioluminescentreporter, an autofluorescent reporter, a chemiluminescent reporter, aluminescent reporter, a colorimetric reporter, or any combinationthereof. Autofluorescent reporters include GFP, mCherry, or derivativesthereof. Colorimetric reporters include pigment-producing enzymes suchas β-galactosidase (e.g. in combination with administration of X-gal),and tyrosinase. Bioluminescent, chemiluminescent or luminescentreporters include luciferases (e.g. in combination with administrationof coelenterazines described herein), including Gaussia luciferases,Renilla luciferases, and Photinus luciferases (e.g. including theengineered Ppy RE8 and RE9 versions described in Branchini et al. Anal.Biochem. 396(2010): 290-297). Reporters detectable by photoacousticimaging include the pigment-producing enzymes such as β-galactosidase(e.g. in combination with administration of X-gal) and tyrosinase,autofluorescent proteins (e.g. GFP, mCherry, or derivatives thereof),non-fluorescent GFP-like chromoproteins (e.g. aeCP597 and cjBlue andderivatives thereof), bacteriophytochrome-based near-infraredfluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713,iRFP720, iRFP713/V256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry,GAF-FP, BphP1-FP/C20S, or AphB variants), and reversibly photoswitchableproteins (e.g. Dronpa, Dronpa-M159T, and BphP1 or variants thereof). Thedetectable nucleic acid biomarker may be a ribozyme, a self-splicingintron, an RNA hairpin, a microRNA, or barcoded versions thereof, orother types of quantifiable RNA. The quantifiable nucleic acid maycomprise a unique sequence detectable by quantitative PCR orhybridization-based techniques. The reporter gene may encode apolypeptide detectable by a non-invasive imaging method. Suchnon-invasive imagine methods include MRI imaging, PET imaging, SPECTimaging, photoacoustic imaging, and bioluminescent imaging. Polypeptidesdetectable by MRI imaging include polypeptide contrast agents, such asferritin (or mutants thereof, such as Pyrococcus furiousus ferritinmutants L55P, F57S, or F123S), or lanthanide-binding proteins (orengineered fusions thereof, such as the LBT-ubiquitin fusions describedin Daughtry et al. ChemBioChem 2012, 13, 2567-2574). Polypeptidesdetectable by PET or SPECT imaging include the human sodium iodidesymporter (e.g. in conjunction with administration of PET-activeiodine/iodide isotopes, see e.g. Penheiter et al. Curr Gene Ther. 2012February; 12(1): 33-47), HSV-tk or mutants thereof such as HSV-sr39tk(e.g. in conjunction with administration of positron-labeledacycloguanosine or pyrimidine analog PET reporters such as [18F]FHBG,see Yaghoubi S S et al. Nat Protoc. 2006; 1(6):3069-75), and thedopamine D2 receptor or mutants thereof such as D2R80A or D2R194A (e.g.in conjunction with administration of positron-labeled D2 binders suchas 3-(2′-[18F]-fluoroethyl)-spiperone). Polypeptides detectable byphotoacoustic imaging include the pigment-producing enzymes such asβ-galactosidase (e.g. in combination with administration of X-gal) andtyrosinase, autofluorescent proteins (e.g. GFP, mCherry, or derivativesthereof), non-fluorescent GFP-like chromoproteins (e.g. aeCP597 andcjBlue and derivatives thereof), bacteriophytochrome-based near-infraredfluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713,iRFP720, iRFP713N256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry,GAF-FP, BphP1-FP/C20S, or AphB variants), and reversibly photoswitchableproteins (e.g. Dronpa, Dronpa-M159T, and BphP1 or variants thereof).Polypeptides detectable by bioluminescent imaging include luciferases(e.g. in combination with administration of coelenterazines describedherein), including Gaussia luciferases, Renilla luciferases, andPhotinus luciferases (e.g. including the engineered Ppy RE8 and RE9versions described in Branchini et al. Anal. Biochem. 396(2010):290-297). In some embodiments, the Polypeptides may be a contrast agent,an enzyme producing a detectable molecule, or a transporter drivingaccumulation of a detectable molecule.

In some cases, the disease affecting the diseased cells may be cancer.Exemplary cancers include, but are not limited to, carcinomas, sarcomas,lymphomas, leukemias, and adenomas. Carcinomas may arise from cells thatcover internal and external parts of the body such as the lung, breast,and colon. Sarcomas may arise from cells that are located in bone,cartilage, fat, connective tissue, muscle, and other supportive tissues.Lymphomas may arise in the lymph nodes and immune system tissues.Leukemias may arise in the bone marrow and accumulate in thebloodstream. Adenomas may arise in the thyroid, the pituitary gland, theadrenal gland, and other glandular tissues. Specific exemplary examplesof cancer types include suitable for detection with the methodsaccording to the disclosure include acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basalcell carcinoma, bile duct cancer, bladder cancer, bone cancers, braintumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignantglioma, ependymoma, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, breastcancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknownprimary origin, central nervous system lymphoma, cerebellar astrocytoma,cervical cancer, childhood cancers, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic myeloproliferative disorders,colon cancer, cutaneous T-cell lymphoma, desmoplastic small round celltumor, endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma, germ cell tumors, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor,gliomas, hairy cell leukemia, head and neck cancer, heart cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer,intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidneycancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, livercancer, lung cancers, such as non-small cell and small cell lung cancer,lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytomaof bone/osteosarcoma, medulloblastoma, melanomas, mesothelioma,metastatic squamous neck cancer with occult primary, mouth cancer,multiple endocrine neoplasia syndrome, myelodysplastic syndromes,myeloid leukemia, nasal cavity and paranasal sinus cancer,nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-smallcell lung cancer, oral cancer, oropharyngeal cancer,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer,ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer,pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonaryblastoma, plasma cell neoplasia, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renalpelvis and ureter transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skincarcinoma merkel cell, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer,thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor(gestational), cancers of unknown primary site, urethral cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia,and Wilms tumor.

In some aspects, the present disclosure provides for a method ofdetecting a disease in a subject comprising an engineered nucleic acidencoding an expressible reporter gene that exhibits about 10% or lessexpression in normal cells versus cells affected by the disease from thesubject when compared to a recombinant nucleic acid comprising areporter gene comprising a nucleic acid sequence of SEQ ID NO: 1 or SEQID NO: 2. The subject may be suspected of having cancer. The disease maybe cancer or any of the subtypes mentioned herein.

In some cases, the engineered nucleic acid may be administeredintravenously, subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally,inhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-175). In someembodiments, the engineered nucleic acid may be administered into atleast one of the cervical, epitrochlear, supraclavicular, cervical,axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, orpopliteal lymph nodes. In some cases, lymph-node based administrationmay serve as a method of centralized local delivery to a tissue region.

In some cases, the method may comprise isolating a biological samplefrom the subject. The biological sample may be a sample collected by anon-invasive method from the subject. Exemplary non-invasive samplesinclude, but are not limited to, samples comprised of naturally shedbodily substances or non-destructive scraping of externally accessibletissues, such as saliva, sputum, sweat, urine, stool, semen, mucus,cervicovaginal secretions, breast milk, rheum, tears, and cheekepithelial swabs. The biological sample may be a sample collected by aminimally-invasive method from the subject. Exemplary minimally-invasivesamples include, but are not limited to, blood samples or fractionsthereof (e.g. obtained by venipuncture or capillary tube), pleural fluidsamples (e.g. obtained by thoracentesis), amniotic fluid samples (e.g.obtained by amniocentesis), and gastric fluid samples (e.g. obtained bygastric lavage). The biological sample may be a sample obtained bybiopsy, such as a skin biopsy sample (e.g. obtained by punch, shave,saucerization, wedge, incisional, or excisional biopsy), a bone marrowsample (e.g. obtained by aspiration biopsy), a lymph node or breastbiopsy (e.g. obtained by fine-needle aspiration, core needle biopsy,vacuum assisted biopsy, or image-guided biopsy), a surgical biopsysample (e.g. of an internal organ obtained by excisional or incisionalbiopsy), or a mouth, GI-tract, lung, bladder, or urinary tract biopsy(e.g. obtained by endoscopy). In some cases, the biological sample maybe collected period of time after the composition is administered to thesubject.

The population of cells may be cultured for least about 15 minutes, atleast about 30 minutes, at least about 1 hour, at least about 2 hours,at least about 4 hours, at least about 8 hours, at least about 16 hours,at least about 24 hours, at least about 36 hours, at least about 48hours, at least about 3 days, at least about 4 days, at least about 5days, at least about 6 days, at least about 7 days, at least about 8days, at least about 9 days, at least about 10 days, at least about 11days, at least about 12 days, at least about 13 days, at least about 14days, at least about 15 days, or at least about 1 month after deliveryof the genetic construct to the cells. The population of cells may becultured for most about 15 minutes, at most about 30 minutes, at mostabout 1 hour, at most about 2 hours, at most about 4 hours, at mostabout 8 hours, at most about 16 hours, at most about 24 hours, at mostabout 36 hours, at most about 48 hours, at most about 3 days, at mostabout 4 days, at most about 5 days, at most about 6 days, at most about7 days, at most about 8 days, at most about 9 days, at most about 10days, at most about 11 days, at most about 12 days, at most about 13days, at most about 14 days, at most about 15 days, or at most about 1month after delivery of the genetic construct to the cells.

In some cases, the method may comprise detecting the polypeptide ornucleic acid sequence. The detecting may comprise a photoacoustic, abioluminescent, fluorescent reporter, chemiluminescent, luminescent,colorimetric or nucleic acid assay. The detecting may also comprise animmunoassay. Immunoassays include those described in e.g., U.S. Pat.Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792. Immunoassays include various sandwich, competitive, ornon-competitive assay formats, which generate a signal that is relatedto the presence or amount of a protein analyte of interest. Any suitableimmunoassay may be utilized, for example, lateral flow, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

The method of detection may comprise sequencing. Sequencing methods mayinclude: Next Generation sequencing, high-throughput sequencing,pyrosequencing, classic Sanger sequencing methods,sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (LifeTechnologies/Thermo-Fisher), massively-parallel sequencing, clonalsingle molecule Array (Solexa), shotgun sequencing, Maxim-Gilbertsequencing, and primer walking.

The detection may comprise a “real time amplification” method also knownas quantitative PCR (qPCR) or Taqman (see, e.g., U.S. Pat. No. 5,210,015to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No.5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362(1995)). The basis for this method of monitoring the formation ofamplification product is to measure continuously PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe. Theprobe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye. The probe is designed tohave at least substantial sequence complementarity with a site on thetarget mRNA or nucleic acid derived from. Upstream and downstream PCRprimers that bind to flanking regions of the locus are also added to thereaction mixture. When the probe is intact, energy transfer between thetwo fluorophores occurs and the quencher quenches emission from thereporter. During the extension phase of PCR, the probe is cleaved by the5′ nuclease activity of a nucleic acid polymerase such as Taqpolymerase, thereby releasing the reporter from thepolynucleotide-quencher and resulting in an increase of reporteremission intensity which can be measured by an appropriate detector. Therecorded values can then be used to calculate the increase in normalizedreporter emission intensity on a continuous basis and ultimatelyquantify the amount of the mRNA being amplified.

In some embodiments, for qPCR or Taqman detection, an RT-PCR step may befirst performed to generate cDNA from cellular RNA. Such amplificationby RT-PCR can either be general (e.g. amplification with partially/fullydegenerate oligonucleotide primers) or targeted (e.g. amplification witholigonucleotide primers directed against specific genes which are to beanalyzed at a later step).

In some embodiments, qPCR or Taqman may be used immediately following areverse-transcriptase reaction performed on isolated cellular mRNA; thisvariety serves to quantitate the levels of individual mRNAs during qPCR.

In some embodiments, for qPCR or Taqman detection or RNA sequencing, a“pre-amplification” step may be first performed on cDNA transcribed fromcellular RNA. This serves to increase signal in conditions where thenatural level of the RNA/cDNA to be detected is very low. Suitablemethods for pre-amplification include but are not limited LM-PCR, PCRwith random oligonucleotide primers (e.g. random hexamer PCR), PCR withpoly-A specific primers, and any combination thereof. Thepre-amplification may be either general or targeted in the same way asthe reverse-transcription reaction described above.

Synthetic Biomarker Design and Method Utilizing miRNA Binding Sites Usedto Modulate Marker Expression

In some aspects, the present disclosure provides for a composition thatexhibits about 10% or less expression in normal cells versus diseasedcells and comprises a recombinant nucleic acid comprising a nucleic acidsequence encoding a reporter gene that includes one or more miRNAbinding sequences in a 3′ untranslated region of the reporter gene.

In some cases, binding or lack of binding of a miRNA expressed in thediseased cells to at least one of the one or more miRNA bindingsequences may result in differential translation or half-life of an mRNAencoding the reporter gene. In some cases, binding of a miRNA expressedin the diseased cells to at least one of the one or more miRNA bindingsequences may result in reduced translation of the reporter gene orreduction of a half-life of an mRNA encoding the reporter gene. In somecases, the reporter gene exhibits increased expression in the cancercell due to downregulation of at least one miRNA expressed in the cancercell.

In some cases, the diseased cell may be a cancerous cell, a cellindicative of an autoimmune disease (e.g. a T-cell or lymphocyte withself-directed activity, or a normal cell damaged by autoimmunity), or acell indicative of a neurodegenerative disease (e.g. a cell bearing atoxic amyloid or proximal to a toxic amyloid). Cancers,neurodegenerative diseases, and autoimmune diseases include any of thediseases described herein. In some cases, the diseased cell may be avirally-infected cell. Exemplary viruses include, but are not limitedto, HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus,herpesviruses, Epstein-Barr virus, cytomegalovirus, and humanT-lymphotropic virus type III.

In some cases, the composition may comprise more than one miRNA bindingsequences in the 3′ untranslated region of the reporter gene. Thecomposition may comprise at least two miRNA binding sequences in the 3′untranslated region of the reporter gene, wherein two miRNA bindingsequences have a substantially identical nucleotide sequence capable ofbinding to a same miRNA. The composition may comprise at least two miRNAbinding sequences in the 3′ untranslated region of the reporter gene,wherein the at least two miRNA binding sequences have differentnucleotide sequences, each of the different nucleotide sequences capableof binding to different miRNAs. The composition may comprise at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 miRNA binding sequences capable of binding the same miRNAor a combination of miRNAs.

In some cases, the recombinant nucleic acid may comprise DNA. When therecombinant nucleic acid comprises DNA, the recombinant nucleic acid maybe part of any of the vectors described herein.

The recombinant nucleic acid may be a synthetic or in vitro-transcribedmRNA.

The one or more miRNA binding sequences may comprise at least onemiR-15, miR-16, let-7, miR-122 or miR-34 binding sequence.

In some cases, the reporter gene may encode a detectable polypeptide ora detectable nucleic acid. The detectable nucleic acid may be aribozyme, a self-splicing intron, an RNA hairpin, a microRNA, RNAaptamers or barcoded versions thereof, or other types of quantifiableRNA. The quantifiable nucleic acid may comprise a unique sequencedetectable by quantitative PCR or hybridization-based techniques.

The reporter gene may encode a photoacoustic reporter, a bioluminescentreporter, an autofluorescent reporter, a chemiluminescent reporter, aluminescent reporter, a colorimetric reporter, or any combinationthereof. Autofluorescent reporters include GFP, mCherry, or derivativesthereof. Colorimetric reporters include pigment-producing enzymes suchas β-galactosidase (e.g. in combination with administration of X-gal),and tyrosinase. Bioluminescent, chemiluminescent or luminescentreporters include luciferases (e.g. in combination with administrationof coelenterazines described herein), including Gaussia luciferases,Renilla luciferases, and Photinus luciferases (e.g. including theengineered Ppy RE8 and RE9 versions described in Branchini et al. Anal.Biochem. 396(2010): 290-297). Reporters detectable by photoacousticimaging include the pigment-producing enzymes such as β-galactosidase(e.g. in combination with administration of X-gal) and tyrosinase,autofluorescent proteins (e.g. GFP, mCherry, or derivatives thereof),non-fluorescent GFP-like chromoproteins (e.g. aeCP597 and cjBlue andderivatives thereof), bacteriophytochrome-based near-infraredfluorescent proteins (e.g. IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713,iRFP720, iRFP713/V256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry,GAF-FP, BphP1-FP/C20S, or AphB variants), and reversibly photoswitchableproteins (e.g. Dronpa, Dronpa-M159T, and BphP1 or variants thereof). Thedetectable nucleic acid may be a ribozyme, a self-splicing intron, anRNA hairpin, a microRNA, or barcoded versions thereof, or other types ofquantifiable RNA. The quantifiable nucleic acid may comprise a uniquesequence detectable by quantitative PCR or hybridization-basedtechniques.

The reporter gene may encode a polypeptide detectable by a non-invasiveimaging method. Such non-invasive imagine methods include MRI imaging,PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescentimaging. Polypeptides detectable by MRI imaging include polypeptidecontrast agents, such as ferritin (or mutants thereof, such asPyrococcus furiousus ferritin mutants L55P, F57S, or F123S), orlanthanide-binding proteins (or engineered fusions thereof, such as theLBT-ubiquitin fusions described in Daughtry et al. ChemBioChem 2012, 13,2567-2574). Polypeptides detectable by PET or SPECT imaging include thehuman sodium iodide symporter (e.g. in conjunction with administrationof PET-active iodine/iodide isotopes, see e.g. Penheiter et al. CurrGene Ther. 2012 February; 12(1): 33-47), HSV-tk or mutants thereof suchas HSV-sr39tk (e.g. in conjunction with administration ofpositron-labeled acycloguanosine or pyrimidine analog PET reporters suchas [18F]FHBG, see Yaghoubi S S et al. Nat Protoc. 2006; 1(6):3069-75),and the dopamine D2 receptor or mutants thereof such as D2R80A orD2R194A (e.g. in conjunction with administration of positron-labeled D2binders such as 3-(2′-[18F]-fluoroethyl)-spiperone). Polypeptidesdetectable by photoacoustic imaging include the pigment-producingenzymes such as β-galactosidase (e.g. in combination with administrationof X-gal) and tyrosinase, autofluorescent proteins (e.g. GFP, mCherry,or derivatives thereof), non-fluorescent GFP-like chromoproteins (e.g.aeCP597 and cjBlue and derivatives thereof), bacteriophytochrome-basednear-infrared fluorescent proteins (e.g. IFP1.4, Wi-Phy IFP1.4rev,IFP2.0, iRFP713, iRFP720, iRFP713N256C, iRFP682, iRFP702, iRFP670, mIFP,iBlueberry, GAF-FP, BphP1-FP/C20S, or AphB variants), and reversiblyphotoswitchable proteins (e.g. Dronpa, Dronpa-M159T, and BphP1 orvariants thereof). Polypeptides detectable by bioluminescent imaginginclude luciferases (e.g. in combination with administration ofcoelenterazines described herein), including Gaussia luciferases,Renilla luciferases, and Photinus luciferases (e.g. including theengineered Ppy RE8 and RE9 versions described in Branchini et al. Anal.Biochem. 396(2010): 290-297). In some embodiments, the polypeptide maybe a contrast agent, an enzyme producing a detectable molecule, or atransporter driving accumulation of a detectable molecule.

In some aspects, the present disclosure provides for a method ofdetecting diseased cells, comprising administering to a subject acomposition that exhibits about 10% or less expression in normal cellsversus diseased cells and comprises a recombinant nucleic acidcomprising a nucleic acid sequence encoding a reporter gene thatincludes one or more miRNA binding sequences in a 3′ untranslated regionof the reporter gene.

In instances where the reporter gene is a polypeptide biomarkerdetectable by a non-invasive imaging method, the method involvingadministering to a subject a composition inducing expression of asynthetic biomarker in a diseased cell may further comprise localizingthe diseased cell in the body of the subject. The localizing may beassociated with a particular resolution, for example 10 mm to 10 cm, atleast 10 mm, or at most 10 cm. The localizing may be associated with aparticular minimum detectable tumor size, for example a tumor sizebetween 3 mm3 and 10 cm3. In some cases, the particular minimum rangemay be may be 1 cm³ to 10 cm³, or 900 mm³ to 1 cm³, or 800 mm³ to 900mm³, or 700 mm³ to 800 mm³, or 600 mm³ to 700 mm³, or 500 mm³ to 600mm³, or 400 mm³ to 500 mm³, or 300 mm³ to 400 mm³, or 200 mm³ to 300mm³, or 100 mm³ to 200 mm³, or 50 mm³ to 100 mm³, or 10 mm³ to 50 mm³,or 3 mm³ to 10 mm³ in size. In some cases, the localization occurs in anon-invasive imaging scan (e.g. PET, MRI, SPECT, etc). In some cases,the localization occurs during surgical intervention in situ, forexample by the use of visual inspection (in the case of visual-rangeabsorbing reporters) or by the use of visual inspection combined withfluorescent excitation.

In some cases, the additional localization step above may be followed bya surgical step to eliminate the detected and/or localized diseasedcell. The surgical step may be performed by the same or different partyto that which administers the biomarker-encoding composition and/orlocalizes the diseased cell. The surgical step may be surgical excisionof the diseased cell or a tumor associated with the diseased cell. Thesurgical or nonsurgical elimination step may involve aminimally-invasive killing technique, such as a radiosurgery (includingbut not limited to Gamma Knife, Reflexion, CyberKnife, and relatedtechniques using targeted ionizing radiation to kill diseased cells).

In some cases, the engineered nucleic acid may be administeredintravenously, subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally, byinhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-175). In someembodiments, the engineered nucleic acid may be administered into atleast one of the cervical, epitrochlear, supraclavicular, cervical,axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, orpopliteal lymph nodes. In some cases, lymph-node based administrationmay serve as a method of centralized local delivery to a tissue region.

In some cases, the method may comprise isolating a biological samplefrom the subject. The biological sample may be a sample collected by anon-invasive method from the subject. Exemplary non-invasive samplesinclude, but are not limited to, samples comprised of naturally shedbodily substances or non-destructive scraping of externally accessibletissues, such as saliva, sputum, sweat, urine, stool, semen, mucus,cervicovaginal secretions, breast milk, rheum, tears, and cheekepithelial swabs. The biological sample may be a sample collected by aminimally-invasive method from the subject. Exemplary minimally-invasivesamples include, but are not limited to, blood samples or fractionsthereof (e.g. obtained by venipuncture or capillary tube), pleural fluidsamples (e.g. obtained by thoracentesis), amniotic fluid samples (e.g.obtained by amniocentesis), and gastric fluid samples (e.g. obtained bygastric lavage). The biological sample may be a sample obtained bybiopsy, such as a skin biopsy sample (e.g. obtained by punch, shave,saucerization, wedge, incisional, or excisional biopsy), a bone marrowsample (e.g. obtained by aspiration biopsy), a lymph node or breastbiopsy (e.g. obtained by fine-needle aspiration, core needle biopsy,vacuum assisted biopsy, or image-guided biopsy), a surgical biopsysample (e.g. of an internal organ obtained by excisional or incisionalbiopsy), or a mouth, GI-tract, lung, bladder, or urinary tract biopsy(e.g. obtained by endoscopy). In some cases, the biological sample maybe collected period of time after the composition is administered to thesubject.

The population of cells may be cultured for least about 15 minutes, atleast about 30 minutes, at least about 1 hour, at least about 2 hours,at least about 4 hours, at least about 8 hours, at least about 16 hours,at least about 24 hours, at least about 36 hours, at least about 48hours, at least about 3 days, at least about 4 days, at least about 5days, at least about 6 days, at least about 7 days, at least about 8days, at least about 9 days, at least about 10 days, at least about 11days, at least about 12 days, at least about 13 days, at least about 14days, at least about 15 days, or at least about 1 month after deliveryof the genetic construct to the cells. The population of cells may becultured for most about 15 minutes, at most about 30 minutes, at mostabout 1 hour, at most about 2 hours, at most about 4 hours, at mostabout 8 hours, at most about 16 hours, at most about 24 hours, at mostabout 36 hours, at most about 48 hours, at most about 3 days, at mostabout 4 days, at most about 5 days, at most about 6 days, at most about7 days, at most about 8 days, at most about 9 days, at most about 10days, at most about 11 days, at most about 12 days, at most about 13days, at most about 14 days, at most about 15 days, or at most about 1month after delivery of the genetic construct to the cells.

In some cases, the method may comprise detecting the polypeptide ornucleic acid sequence. The detecting may comprise a photoacoustic, abioluminescent, fluorescent reporter, chemiluminescent, luminescent,colorimetric or nucleic acid assay. The detecting may also comprise animmunoassay. Immunoassays include those described in e.g., U.S. Pat.Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792. Immunoassays include various sandwich, competitive, ornon-competitive assay formats, which generate a signal that is relatedto the presence or amount of a protein analyte of interest. Any suitableimmunoassay may be utilized, for example, lateral flow, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

The method of detection may comprise sequencing. Sequencing methods mayinclude: Next Generation sequencing, high-throughput sequencing,pyrosequencing, classic Sanger sequencing methods,sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (LifeTechnologies/Thermo-Fisher), massively-parallel sequencing, clonalsingle molecule Array (Solexa), shotgun sequencing, Maxim-Gilbertsequencing, and primer walking.

The detection may comprise a “real time amplification” method also knownas quantitative PCR (qPCR) or Taqman (see, e.g., U.S. Pat. No. 5,210,015to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No.5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362(1995)). The basis for this method of monitoring the formation ofamplification product is to measure continuously PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe. Theprobe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye. The probe is designed tohave at least substantial sequence complementarity with a site on thetarget mRNA or nucleic acid derived from. Upstream and downstream PCRprimers that bind to flanking regions of the locus are also added to thereaction mixture. When the probe is intact, energy transfer between thetwo fluorophores occurs and the quencher quenches emission from thereporter. During the extension phase of PCR, the probe is cleaved by the5′ nuclease activity of a nucleic acid polymerase such as Taqpolymerase, thereby releasing the reporter from thepolynucleotide-quencher and resulting in an increase of reporteremission intensity which can be measured by an appropriate detector. Therecorded values can then be used to calculate the increase in normalizedreporter emission intensity on a continuous basis and ultimatelyquantify the amount of the mRNA being amplified.

In some embodiments, for qPCR or Taqman detection, an RT-PCR step may befirst performed to generate cDNA from cellular RNA. Such amplificationby RT-PCR can either be general (e.g. amplification with partially/fullydegenerate oligonucleotide primers) or targeted (e.g. amplification witholigonucleotide primers directed against specific genes which are to beanalyzed at a later step).

In some embodiments, qPCR or Taqman may be used immediately following areverse-transcriptase reaction performed on isolated cellular mRNA; thisvariety serves to quantitate the levels of individual mRNAs during qPCR.

In some embodiments, for qPCR or Taqman detection or RNA sequencing, a“pre-amplification” step may be first performed on cDNA transcribed fromcellular RNA. This serves to increase signal in conditions where thenatural level of the RNA/cDNA to be detected is very low. Suitablemethods for pre-amplification include but are not limited LM-PCR, PCRwith random oligonucleotide primers (e.g. random hexamer PCR), PCR withpolyA specific primers, and any combination thereof. Thepre-amplification may be either general or targeted in the same way asthe reverse-transcription reaction described above.

CELiD-Based Design and Method for Improved Safety and PersistentSynthetic Biomarker Expression

In some aspects, the present disclosure provides for a compositionexhibiting significantly longer expression of synthetic biomarker versusplasmid DNA or minicircle DNA comprising a linear vector comprising adouble-stranded nucleic acid comprising a promoter operatively linked toa DNA sequence encoding a synthetic biomarker, wherein a forward and areverse strand of the double-stranded nucleic acid are covalently linkedon each of their terminal ends, wherein the promoter induces expressionof the synthetic biomarker in a diseased cell preferentially overexpression of the synthetic biomarker in a non-diseased cell such that arelative concentration of the synthetic biomarker expressed in thediseased cell over the non-diseased cell is greater than 1.0.

In some cases, the disease may be cancer, an autoimmune disease (e.g. aT-cell or lymphocyte with self-directed activity, or a normal celldamaged by autoimmunity), or a neurodegenerative disease (e.g. a cellbearing a toxic amyloid or proximal to a toxic amyloid). Exemplarycancers, autoimmune diseases, and neurodegenerative diseases include anyof those described herein. In some cases, the disease may be a viralinfection. Exemplary viral infections include, but are not limited to,those caused by HIV, hepatitis C virus, hepatitis B virus, hepatitis Dvirus, herpesviruses, Epstein-Barr virus, cytomegalovirus, and humanT-lymphotropic virus type III.

In some cases, the linear vector may comprise inverted terminal repeats(ITRs) flanking the promoter operatively linked to the DNA sequenceencoding the synthetic biomarker, wherein the ITRs are derived from anAdeno-Associated Virus (AAV). In some cases, the AAV may be AAV2. Insome cases, the promoter may drive expression of the synthetic biomarkerselectively in a plurality of diseased cells in a subject.

In some cases, the promoter may have a cell-type specificity. In somecases, the promoter is a pan-cancer specific promoter. In some cases,the promoter may be a cancer-specific promoter. In some cases, thepromoter may be any of the specific promoters mentioned herein.

In some cases, the synthetic biomarker may comprise an MRI reporter, aPET reporter, a SPECT reporter, a photoacoustic reporter, abioluminescent reporter, a fluorescent reporter, chemiluminescentreporter, a luminescent reporter, colorimetric reporter, a quantifiablenucleic acid biomarker and combinations thereof and any combinationthereof. The detectable nucleic biomarker acid may be a ribozyme, aself-splicing intron, an RNA hairpin, a microRNA, or barcoded versionsthereof, or other types of quantifiable RNA. The quantifiable nucleicacid biomarker may comprise a unique sequence detectable by quantitativePCR or hybridization-based techniques.

In some aspects, the present disclosure provides for a method ofidentifying a diseased cell, comprising: (a) administering to a subjecta composition, wherein the composition exhibits significantly longerexpression of synthetic biomarker versus plasmid DNA or minicircle DNAcomprising a linear vector comprising a double-stranded nucleic acidcomprising a promoter operatively linked to a DNA sequence encoding asynthetic biomarker, wherein a forward and a reverse strand of thedouble-stranded nucleic acid are covalently linked on each of theirterminal ends; and (b) detecting the synthetic biomarker, wherein thesynthetic biomarker is expressed in a diseased cell preferentially overexpression of the synthetic biomarker in non-diseased cells in thesubject such that a relative concentration of the synthetic biomarkerexpressed in the diseased cell over the non-diseased cells is greaterthan 1.0.

In instances where the synthetic biomarker is a polypeptide biomarkerdetectable by a non-invasive imaging method, the method involvingadministering to a subject a composition inducing expression of asynthetic biomarker in a diseased cell may further comprise (d)localizing the diseased cell in the body of the subject. The localizingmay be associated with a particular resolution, for example 10 mm to 10cm, at least 10 mm, or at most 10 cm. The localizing may be associatedwith a particular minimum detectable tumor size, for example a tumorsize between 3 mm3 and 10 cm³. In some cases, the particular minimumrange may be may be 1 cm³ to 10 cm³, or 900 mm³ to 1 cm³, or 800 mm³ to900 mm³, or 700 mm³ to 800 mm³, or 600 mm³ to 700 mm³, or 500 mm³ to 600mm³, or 400 mm³ to 500 mm³, or 300 mm³ to 400 mm³, or 200 mm³ to 300mm³, or 100 mm³ to 200 mm³, or 50 mm³ to 100 mm³, or 10 mm³ to 50 mm³,or 3 mm³ to 10 mm³ in size. In some cases, the localization occurs in anon-invasive imaging scan (e.g. PET, MRI, SPECT, etc). In some cases,the localization occurs during surgical intervention in situ, forexample by the use of visual inspection (in the case of visual-rangeabsorbing reporters) or by the use of visual inspection combined withfluorescent excitation.

In some cases, the additional localization step above may be followed bya surgical step to eliminate the detected and/or localized diseasedcell. The surgical step may be performed by the same or different partyto that which administers the biomarker-encoding composition and/orlocalizes the diseased cell. The surgical step may be surgical excisionof the diseased cell or a tumor associated with the diseased cell. Thesurgical or nonsurgical elimination step may involve aminimally-invasive killing technique, such as a radiosurgery (includingbut not limited to Gamma Knife, Reflexion, CyberKnife, and relatedtechniques using targeted ionizing radiation to kill diseased cells).

In some cases, the composition is administered intravenously,subcutaneously, intraventricularly, intrathecally,intracerebroventricularly, transdermally, intramuscularly, orally, byinhalation, nasally, rectally, intratumorally, or proxi-tumorally to thesubject. Proxi-tumorally may denote administration to the tissue withinproximity of a tumor, or administration into a region that would bepredicted to be accessible to the tumor via the lymphatic system (e.g.an adjoining lymph node). Intratumoral or proxi-tumoral approaches mayinvolve the use of additional imaging techniques such as e.g. endoscopicultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013;2013: 207129) or via a brochioscope (see e.g. Rojas-Solano et al. JBronchology Intery Pulmonol. 2018 July; 25(3): 168-175). In someembodiments, the composition is administered into at least one of thecervical, epitrochlear, supraclavicular, cervical, axillary,mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteallymph nodes. In some cases, lymph-node based administration may serve asa method of centralized local delivery to a tissue region.

In some cases, the detection of the diseased cell may have an accuracyat least about 50%, at least about 53%, at least about 55%, at leastabout 57%, at least about 60%, at least about 63%, at least about 65%,at least about 67%, at least about 70%, at least about 72%, at leastabout 75%, at least about 77%, at least about 78%, at least about 79%,at least about 80%, at least about 81%, at least about 82%, 83%, atleast about 84%, 85%, at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, or any range in between these values. Insome cases the detection of the diseased cell may have an accuracy of atmost about 53%, 55%, 57%, 60%, 63%, 65%, 67%, 70%, 72%, 75%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, or any range in between these values.

In some cases, the detection of the diseased cell may have a sensitivityof at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range in betweenthese values. In some cases, the detection of the diseased cell may havea sensitivity of at most about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any rangein between these values.

In some cases, the detection of the diseased cell may have a specificityof at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range in betweenthese values. In some cases, the detection of the diseased cell may havea specificity of at most about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any rangein between these values

In some cases, the detection of the diseased cell may have a negativepredictive value (NPV) of at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.2%, 95.5%, 95.7%,96%, 96.2%, 96.5%, 96.7%, 97%, 97.2%, 97.5%, 97.7%, 98%, 98.2%, 98.5%,98.7%, 99%, 99.2%, 99.5%, 99.7%, or 99.9%, or any range in between thesevalues. In some cases, the detection of the diseased cell may have a NPVof at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 95.2%, 95.5%, 95.7%, 96%, 96.2%, 96.5%, 96.7%, 97%,97.2%, 97.5%, 97.7%, 98%, 98.2%, 98.5%, 98.7%, 99%, 99.2%, 99.5%, 99.7%,or 99.9%, or any range in between these values.

In some cases, the detection of the diseased cell may have a positivepredictive value (PPV) of at least about 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 63%, 65%, 67%, 70%, 72%,75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any rangebetween these values. In some cases, the detection of the diseased cellmay have a PPV of at most about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 60%, 63%, 65%, 67%, 70%, 72%, 75%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range between these values.

In some cases, detection of the diseased cell may involve using anon-invasive imaging method performed on the subject. The non-invasiveimaging method may be photoacoustic, MRI, SPECT, or PET imaging. Theimaging method may be performed at least about 15 minutes, at leastabout 30 minutes, at least about 1 hour, at least about 2 hours, atleast about 4 hours, at least about 8 hours, at least about 16 hours, atleast about 24 hours, at least about 36 hours, at least about 48 hours,at least about 3 days, at least about 4 days, at least about 5 days, atleast about 6 days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, at least about 14 days, atleast about 15 days, at least about 1 month, at least about 2 months, atleast about 3 months, at least about 4 months, at least about 5 months,at least about 6 months, or at least about 1 year after administrationof the composition to the subject. The imaging method may be performedat most about 15 minutes, at most about 30 minutes, at most about 1hour, at most about 2 hours, at most about 4 hours, at most about 8hours, at most about 16 hours, at most about 24 hours, at most about 36hours, at most about 48 hours, at most about 3 days, at most about 4days, at most about 5 days, at most about 6 days, at most about 7 days,at most about 8 days, at most about 9 days, at most about 10 days, atmost about 11 days, at most about 12 days, at most about 13 days, atmost about 14 days, at most about 15 days, at most about 1 month, atmost about 2 months, at most about 3 months, at most about 4 months, atmost about 5 months, at most about 6 months, or at most about 1 yearafter administration of the composition to the subject. In someembodiments, the imaging method may be performed multiple times afteradministration of the composition to the subject (e.g. to monitorsynthetic biomarker levels over time). The imaging method may beperformed at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25times after administration of the composition to the subject. Theimaging method may be performed weekly or monthly following afteradministration of the composition to the subject.

In some cases, detection of the diseased cell may involve detection froma biological sample from the subject. In some cases, the method maycomprise isolating a biological sample from the subject. The biologicalsample may be a sample collected by a non-invasive method from thesubject. Exemplary non-invasive samples include, but are not limited to,samples comprised of naturally shed bodily substances or non-destructivescraping of externally accessible tissues, such as saliva, sputum,sweat, urine, stool, semen, mucus, cervicovaginal secretions, breastmilk, rheum, tears, and cheek epithelial swabs. The biological samplemay be a sample collected by a minimally-invasive method from thesubject. Exemplary minimally-invasive samples include, but are notlimited to, blood samples or fractions thereof (e.g. obtained byvenipuncture or capillary tube), pleural fluid samples (e.g. obtained bythoracentesis), amniotic fluid samples (e.g. obtained by amniocentesis),and gastric fluid samples (e.g. obtained by gastric lavage). Thebiological sample may be a sample obtained by biopsy, such as a skinbiopsy sample (e.g. obtained by punch, shave, saucerization, wedge,incisional, or excisional biopsy), a bone marrow sample (e.g. obtainedby aspiration biopsy), a lymph node or breast biopsy (e.g. obtained byfine-needle aspiration, core needle biopsy, vacuum assisted biopsy, orimage-guided biopsy), a surgical biopsy sample (e.g. of an internalorgan obtained by excisional or incisional biopsy), or a mouth,GI-tract, lung, bladder, or urinary tract biopsy (e.g. obtained byendoscopy).

In some cases, detection may be performed a period of time after thecomposition is administered to the subject. The period of time may be atleast about 15 minutes, at least about 30 minutes, at least about 1hour, at least about 2 hours, at least about 4 hours, at least about 8hours, at least about 16 hours, at least about 24 hours, at least about36 hours, at least about 48 hours, at least about 3 days, at least about4 days, at least about 5 days, at least about 6 days, at least about 7days, at least about 8 days, at least about 9 days, at least about 10days, at least about 11 days, at least about 12 days, at least about 13days, at least about 14 days, at least about 15 days, at least about 1month, at least about 2 months, at least about 3 months, at least about4 months, at least about 5 months, or at least about 6 months after thecomposition is administered to the subject. The period of time may be atmost about 15 minutes, at most about 30 minutes, at most about 1 hour,at most about 2 hours, at most about 4 hours, at most about 8 hours, atmost about 16 hours, at most about 24 hours, at most about 36 hours, atmost about 48 hours, at most about 3 days, at most about 4 days, at mostabout 5 days, at most about 6 days, at most about 7 days, at most about8 days, at most about 9 days, at most about 10 days, at most about 11days, at most about 12 days, at most about 13 days, at most about 14days, at most about 15 days, at most about 1 month, at most about 2months, at most about 3 months, at most about 4 months, at most about 5months, or at most about 6 months after the composition is administeredto the subject. In some embodiments, the biological sample may beobtained, and any biomarker detection protocols performed multiple timesafter the composition is administered to the subject (e.g. to monitorsynthetic biomarker levels over time). Detection from the biologicalsample may occur at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or 25 times after the composition is administered to the subject.Detection from the biological sample may occur weekly or monthly afterthe composition is administered to the subject.

Design and Method for Reduction of Background Biomarker Expression fromNormal Organ Tissues

In some aspects, the present disclosure provides for a compositioncomprising a vector expressing a synthetic biomarker, wherein thesynthetic biomarker exhibits about 10% or less expression in normalorgan cells versus diseased cells. In some cases, the organ may beliver, kidney, spleen, or a combination thereof. In some cases, thevector may comprise a recombinant nucleic acid encoding a promoteroperably linked to a synthetic biomarker.

Promoters may include pan-cancer specific promoters, cancer-specificpromoters, or any of the specific promoters described herein.

Synthetic biomarkers may include MRI reporters, PET reporters, SPECTreporters, photoacoustic reporters, bioluminescent reporters,autofluorescent reporters, chemiluminescent reporters, luminescencereporters, colorimetric reporters, quantifiable nucleic acids and anycombination thereof. Detectable nucleic acids may be ribozymes,self-splicing introns, RNA hairpins, microRNAs, or barcoded versionsthereof, or other types of quantifiable RNA. The quantifiable nucleicacids may comprise a unique sequence detectable by quantitative PCR orhybridization-based techniques.

The vector may comprise regulatory elements that silence or attenuatetranscription or mRNA half-life of the nucleic acid sequence in normalliver, spleen, or kidney cells. The regulatory elements may comprise oneor more miRNA target sequences in a transcribed, but an untranslatedregion, of the recombinant nucleic acid. Presence of the one or moremiRNA target sequences may inhibit expression from the recombinantnucleic acid sequence. One or more miRNA target sequences may compriseat least one miRNA target sequence for at least one tissue specificmiRNA. At least one tissue specific miRNA may comprise at least onemiRNA enriched in normal hepatic, renal, or splenic tissues. At leastone miRNA enriched in normal hepatic tissues may comprise, but not belimited to, miR-122, miR-33, miR-33*, miR-223, miR-30c, miR-144,miR-148a, miR-24, miR-29, or any combination thereof.

The composition may comprise a transfection agent as described herein.

Synthetic or Engineered Cell Design/Method for Use with SyntheticBiomarkers

In some aspects, the present disclosure provides for an engineeredparticle that mimics one or many functions of a biological cell ormacrophage including inducing the expression of a biomarker in adiseased cell preferentially over expression of the biomarker innon-diseased cells such that the relative concentration ratio of thebiomarker expressed in the diseased cell over the non-diseased cells isgreater than 1.0.

The engineered particle may be an artificial cell, a minimal cell, or alipid-enclosed synthetic particle. Production of artificial cells isdescribed in e.g. Bastiaan et al. Acc. Chem. Res., 2017, 50 (4), pp769-777, which is incorporated by reference herein. The engineeredparticle may comprise biological membranes, polymeric membranes, simplepolymers, crosslinked proteins, lipid membranes or polymer-lipidcomplexes formed in vitro or in vivo with purified components intonanoparticles, liposomes, polymersomes, exosomes, microvesicles,apoptotic blebs, transport vesicles, synaptic vesicles, secretoryvesicles, or microcapsules. The engineered particle may comprise atransmembrane chimeric protein or a naturally occurring proteincomprising an extraparticle specific binding domain operably linked toan intraparticle signaling domain, wherein the intraparticle signalingdomain is capable of activating at least one enzymatic reaction withinthe engineered particle. The engineered particle may comprise a nucleicacid sequence encoding a synthetic biomarker, wherein the at least oneenzymatic reaction results in the production of the synthetic biomarkerwithin the engineered particle. Exemplary synthetic biomarkers includeany reporters described herein. The extraparticle specific bindingdomain may comprise an scFv or a Fab fragment. The engineered particlemay comprise cytoplasm and other components isolated from intact cells.The engineered particle may comprise purified recombinant macromolecularcomponents or macromolecular components such as, but not limited to,cellular proteins, DNA, synthetic gene circuits, organelles, ATP,enzymes, NADP, transcription factors, nucleotides, or cell-freetranscription-translation extracts.

Synthetic Biomarker Design and Method Using Multiplex CombinatorialBiomarkers for Disease Detection and/or Generating a Profile of aSubject's Disease

In some aspects, the present disclosure provides for at least onevector, wherein the at least one vector comprises: a plurality ofdifferent promoters operably linked to a plurality of different nucleicacid sequences, wherein the promoters drive expression of the pluralityof nucleic acid sequences in a cell to yield a plurality of polypeptidesor nucleic acid biomarker sequences, wherein the promoters induceexpression of the plurality of polypeptides or nucleic acid biomarkersequences in a diseased cell preferentially over expression of theplurality of polypeptides or nucleic acid biomarker sequences innon-diseased cells in a subject such that a relative ratio of theplurality of polypeptides or nucleic acid biomarker sequences expressedin the diseased cell over the non-diseased cells is greater than 1.0. Insome embodiments, each of said promoters may induce expression of saidplurality of polypeptides or nucleic acid biomarker sequences in adiseased cell preferentially over expression of said plurality ofpolypeptides or nucleic acid biomarker sequences in non-diseased cellsin said subject such that a relative ratio of said plurality ofpolypeptides or nucleic acid biomarker sequences expressed in saiddiseased cell over said non-diseased cells is greater than 1.0.

In some aspects, the present disclosure provides methods for generatinga profile of a subject's disease. The methods comprise contacting one ormore cells of said subject with a plurality of genetic constructions,wherein the plurality of genetic constructs comprises a plurality ofdisease-activated promoters respectively operably linked to a pluralityof barcode molecules and the disease-activated promoter drivesexpression of the corresponding barcode molecule in a cell affected bythe disease. Further, the methods comprise quantifying expression levelsof the plurality of barcode molecules to generate the profile. In someembodiments, the methods further comprise detecting a disease based onthe generated profile, which comprises expression levels of the barcodemolecules corresponding to the plurality of disease-activated promotersusing a classifier (machine learning or classifier algorithm) to detectthe disease or the absence thereof.

By ascribing an exclusive label to a unique member within a largergroup, barcodes afford the opportunity to identify and quantify thatmember (e.g. expression of a reporter under the control of a particularcancer specific promoter) within the context of a larger and morecomplex mixture of many members (e.g. multiple promoter-reporterconstructs expressed within the same cell), as well as offering theopportunity to isolate a single member from the complex mixture. Forinstance, in the case of barcodes based on nucleic acids, hybridizationof barcodes based on base pairing complementarity may be used to captureand isolate or otherwise reduce the complexity of a mixture by saidcapture event. For barcodes based on peptides, unique features includingimmunocapture or interactions of ligands and receptors may be used tocapture and isolate or otherwise reduce the complexity of a mixture bysaid capture event.

The methods further comprise detecting said disease or absence thereofwith an AUC (area under the curve) value of at least 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. The methods furthercomprise detecting said disease or absence thereof with a specificity ofat least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more. The methods further comprise detecting said disease or absencethereof with a sensitivity of at least 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

After quantifying the barcode molecule levels in a first-round screeningand determining a particular disease, for example a particular cancer(i.e., breast cancer), or a cancer within a particular tissue origin, aplurality of genetic constructs comprising the particular cancer (i.e.,breast cancer)-activated promoters may be used ex-vivo or in vivo totransfect one or more cells of the same subject that has gone throughthe first round of screening. The following rounds (e.g., more than two,three, four, five, six, seven, eight, nine, ten or more) may serve toincrease the accuracy of disease identification (including tissue originidentification).

In some embodiments, the methods of contacting one or more cells can beperformed ex-vivo. In some embodiments, the methods of contacting one ormore cells can be performed in-vivo.

In some embodiments, the at least one vector may be a single vectorcontaining all the elements above. In some embodiments, the at least onevector may be a plurality of vectors each comprising a distinctpromoter. In some embodiments, each of the plurality of vectors eachcomprising a distinct promoter also comprise a distinct polypeptide ornucleic acid biomarker sequence. When the at least one vector is aplurality of vectors each comprising a distinct promoter, the vectorsmay be administered within at least 8, at least 12, at least 16, atleast 24, at least 32, at least 48, at least 56, or at least 72 hours ofeach other. When the at most one vector is a plurality of vectors eachcomprising a distinct promoter, the vectors may be administered withinat most 8, at most 12, at most 16, at most 24, at most 32, at most 48,at most 56, or at most 72 hours of each other.

In some embodiments, the distinct promoter is a disease-activatedpromoter. In some embodiments, the disease-activated promoter is acancer-activated promoter as disclosed herein. In some embodiments, theplurality of cancer-activated promoters comprises promoters activated ina plurality of cancers within different tissue origins. For example, theplurality of cancer-activated promoters comprises a first promoteractivated in a lung cancer within lung tissue, a second promoteractivated in liver cancer within liver tissue, a third promoteractivated in breast cancer within breast tissue, a fourth promoteractivated in pancreatic cancer within pancreas tissue, etc. In someembodiments, the plurality of cancer-specific promoters are activated ina plurality (e.g. two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore, eleven or more, twelve or more, thirteen or more, fourteen ormore, fifteen or more, sixteen or more, seventeen or more, eighteen ormore, nineteen or more, twenty or more, twenty-five or more, or thirtyor more) of different tissue origins.

In some embodiments, the plurality of cancer-specific promoterscomprises a first promoter that produces a strong signal in detectionafter being activated in one or more different types of cancers. Forexample, promoter MMP11 produces strong signals in detection after beingactivated in cancers, such as BLCA, BRCA, CESE, CHOL, COAD, ESCA, HNSC,LUAD, LUSC, PAAD, OV, READ, STAD, SARC, etc. In some embodiments, theplurality of cancer-specific promoters comprises a second promoter thatproduces a low background signal. For example, promoter MMP13 producesalmost non-existing signal in detection in cancer types that MMP13 isnot activated. In some embodiments, the plurality of cancer-specificpromoters comprises a third promoter that has a highsignal-to-background signal ratio. For example, promoter MMP12 producesa sufficient amount of signal in detection after being activated inparticular cancer types that MMP12 can be activated, and at the sametime, produces low signal in detection in particular cancer types thatMMP12 are not designed to be activated. In certain embodiments, theplurality of cancer-specific promoters comprise all three types ofpromoter as disclosed herein. In certain embodiments, the plurality ofcancer-specific promoters comprises one or more promoters with highsignals in detection and one or more promoters with low backgroundsignals in detection. In certain embodiments, plurality ofcancer-specific promoters comprises one or more promoters with highsignals in detection and one or more promoters with highsignal-to-background signal ratio. In certain embodiments, the pluralityof cancer-specific promoters comprises one or more promoters with lowbackground signals in detection and one or more promoters with highsignal-to-background signal ratio.

In certain embodiments, the plurality of disease-activated promoterscomprises a plurality of cancer-activated promoters that are selectivefor and activated in a selected group of tissue origin. For example, theplurality of disease-activated promoters comprises one or more promotersthat are selective for and activated in multiple tissues, such asbreast, lung, liver, etc. In some embodiments, the plurality ofdisease-activated promoters comprises a plurality of cancer-activatedpromoters that are selective for and activated in the same tissueorigin. For instances, the plurality of disease-activated promoterscomprises several different promoters and each of them is selective forand activated in the same tissue, such as breast tissue.

In certain embodiments, the plurality of disease-activated promoterscomprises a plurality of cancer-activated promoters activated in aplurality of different molecular subtypes of a cancer respectivelywithin the same tissue origin. For example, the plurality ofdisease-activated promoters comprises a plurality of disease-specificpromoters activated in luminal A breast cancer, luminal B breast cancer,triple-negative/basal-like breast cancer, HER2-enriched breast cancer,and/or normal-like breast cancer. The plurality of disease-activatedpromoters comprises a plurality of disease-specific promoters activatedin CMS1, CMS2, CMS3, and/or CMS4 colorectal cancer. In certainembodiments, the plurality of disease-activated promoters comprises aplurality of cancer-activated promoters activated in one molecularsubtype of a cancer within a tissue origin. For example, the pluralityof disease-activated promoters comprises more than one differentpromoters that are activated in luminal A breast cancer. In certainembodiments, the plurality of disease-activated promoters comprises twoor more different cancer-activated promoters activated in one stage of acancer within a tissue origin. In certain embodiments, the plurality ofdisease-activated promoters comprises disease-specific promoters thatare activated in different stages of a cancer with a molecular subtypewithin a tissue origin.

In some cases, the disease is cancer, an autoimmune disease (e.g. aT-cell or lymphocyte with self-directed activity, or a normal celldamaged by autoimmunity), or a neurodegenerative disease (e.g. a cellbearing a toxic amyloid or proximal to a toxic amyloid). Exemplarycancers include, but are not limited to, carcinomas, sarcomas,lymphomas, leukemias, and adenomas. Carcinomas may arise from cells thatcover internal and external parts of the body such as the lung, breast,and colon. Sarcomas may arise from cells that are located in bone,cartilage, fat, connective tissue, muscle, and other supportive tissues.Lymphomas may arise in the lymph nodes and immune system tissues.Leukemias may arise in the bone marrow and accumulate in thebloodstream. Adenomas may arise in the thyroid, the pituitary gland, theadrenal gland, and other glandular tissues. Specific exemplary examplesof cancer types include suitable for detection with the methodsaccording to the disclosure include acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basalcell carcinoma, bile duct cancer, bladder cancer, bone cancers, braintumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignantglioma, ependymoma, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, breastcancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknownprimary origin, central nervous system lymphoma, cerebellar astrocytoma,cervical cancer, childhood cancers, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic myeloproliferative disorders,colon cancer, cutaneous T-cell lymphoma, desmoplastic small round celltumor, endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma, germ cell tumors, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor,gliomas, hairy cell leukemia, head and neck cancer, heart cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer,intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidneycancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, livercancer, lung cancers, such as non-small cell and small cell lung cancer,lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytomaof bone/osteosarcoma, medulloblastoma, melanomas, mesothelioma,metastatic squamous neck cancer with occult primary, mouth cancer,multiple endocrine neoplasia syndrome, myelodysplastic syndromes,myeloid leukemia, nasal cavity and paranasal sinus cancer,nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-smallcell lung cancer, oral cancer, oropharyngeal cancer,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer,ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer,pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonaryblastoma, plasma cell neoplasia, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renalpelvis and ureter transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skincarcinoma merkel cell, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer,thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor(gestational), cancers of unknown primary site, urethral cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia,Wilms tumor, Acute Myeloid Leukemia, Adrenocortical Carcinoma, BladderUrothelial Carcinoma, Breast Ductal Carcinoma, Breast Lobular Carcinoma,Cervical Carcinoma, Cholangiocarcinoma, Colorectal Adenocarcinoma,Esophageal Carcinoma, Gastric Adenocarcinoma, Glioblastoma Multiforme,Head and Neck Squamous Cell Carcinoma, Hepatocellular Carcinoma, KidneyChromophobe Carcinoma, Kidney Clear Cell Carcinoma, Kidney PapillaryCell Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma, Lung SquamousCell Carcinoma, Mesothelioma, Ovarian Serous Adenocarcinoma, PancreaticDuctal Adenocarcinoma, Paraganglioma & Pheochromocytoma, ProstateAdenocarcinoma, Sarcoma, Skin Cutaneous Melanoma, Testicular Germ CellCancer, Thymoma, Thyroid Papillary Carcinoma, Uterine Carcinosarcoma,Uterine Corpus Endometrioid Carcinoma, Uveal Melanoma, lip melanoma,spindle cell carcinoma, liposarcoma, nasal sarcoma, mammaryadenocarcinoma, insulinoma, osteosarcoma, mast cell tumors,hemangiosarcoma, non-small cell lung carcinoma (NSCLC), marginallymphoma, malignant melanoma, or chronic lymphocytic leukemia.

In some cases, the disease may be a viral infection. Exemplary viralinfections include, but are not limited to, those caused by HIV,hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesviruses,Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus typeIII.

In some cases, the disease may be an autoimmune disease. Exemplaryautoimmune diseases include, but are not limited to, Achalasia,Addison's disease, Adult Still's disease, Agammaglobulinemia, Alopeciaareata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmunedysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis,Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmuneoophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmuneretinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN),Baló disease, Behcet's disease, Benign mucosal pemphigoid, Bullouspemphigoid, Castleman disease (CD), Celiac disease, Chagas disease,Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronicrecurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS)or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan'ssyndrome, Cold agglutinin disease, Congenital heart block, Coxsackiemyocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis,Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus,Dressler's syndrome, Endometriosis, Eosinophilic esophagitis (EoE),Eosinophilic fasciitis, Erythema nodosum, Essential mixedcryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis,Giant cell arteritis (temporal arteritis), Giant cell myocarditis,Glomerulonephritis, Goodpasture's syndrome, Granulomatosis withPolyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto'sthyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpesgestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa(HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy,IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP),Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenilearthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM),Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis,Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgAdisease (LAD), Lupus, Lyme disease chronic, Meniere's disease,Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy(MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis,Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocularcicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR),PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis(peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheralneuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMSsyndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III,Polymyalgia rheumatica, Polymyositis, Postmyocardial infarctionsyndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis,Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis,Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum,Raynaud's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy,Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitonealfibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidtsyndrome, Scleritis, Scleroderma, Sjögren's syndrome, Sperm & testicularautoimmunity, Stiff person syndrome (SPS), Subacute bacterialendocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transversemyelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiatedconnective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, andVogt-Koyanagi-Harada Disease.

In some cases, the disease may be a neurodegenerative disease.Neurodegenerative diseases include, but are not limited to, Multiplesclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), andAmyotrophic lateral sclerosis (ALS), or neurodegeneration due toinfection by viruses of families Herpesviridae, Polyomaviridae,Bornaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae,Flaviviridae, Picornaviridae, or Retroviridae (see Zhou et al. Virol J.2013; 10: 172).

In some cases, the disease may be a viral infection. Exemplary viralinfections include, but are not limited to, those caused by HIV,hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesviruses,Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus typeIII.

In some cases, at least one of the plurality of polypeptides or nucleicacid biomarker sequences may comprise a sequence of a polypeptidedetectable by non-invasive imaging. Such non-invasive imagine methodsinclude MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging,and bioluminescent imaging. Synthetic biomarker polypeptides detectableby MRI imaging include polypeptide contrast agents, such as ferritin (ormutants thereof, such as Pyrococcus furiousus ferritin mutants L55P,F57S, or F123S), or lanthanide-binding proteins (or engineered fusionsthereof, such as the LBT-ubiquitin fusions described in Daughtry et al.ChemBioChem 2012, 13, 2567-2574). Synthetic biomarkers detectable by PETor SPECT imaging include the human sodium iodide symporter (e.g. inconjunction with administration of PET-active iodine/iodide isotopes,see e.g. Penheiter et al. Curr Gene Ther. 2012 February; 12(1): 33-47),HSV-tk or mutants thereof such as HSV-sr39tk (e.g. in conjunction withadministration of positron-labeled acycloguanosine or pyrimidine analogPET reporters such as [18F]FHBG, see Yaghoubi S S et al. Nat Protoc.2006; 1(6):3069-75), and the dopamine D2 receptor or mutants thereofsuch as D2R80A or D2R194A (e.g. in conjunction with administration ofpositron-labeled D2 binders such as 3-(2′-[18F]-fluoroethyl)-spiperone).Synthetic biomarkers detectable by photoacoustic imaging include thepigment-producing enzymes such as β-galactosidase (e.g. in combinationwith administration of X-gal) and tyrosinase, autofluorescent proteins(e.g. GFP, mCherry, or derivatives thereof), non-fluorescent GFP-likechromoproteins (e.g. aeCP597 and cjBlue and derivatives thereof),bacteriophytochrome-based near-infrared fluorescent proteins (e.g.IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713/V256C,iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphP1-FP/C20S, orAphB variants), and reversibly photoswitchable proteins (e.g. Dronpa,Dronpa-M159T, and BphP1 or variants thereof). Synthetic biomarkersdetectable by bioluminescent imaging include luciferases (e.g. incombination with administration of coelenterazines described herein),including Gaussia luciferases, Renilla luciferases, and Photinusluciferases (e.g. including the engineered Ppy RE8 and RE9 versionsdescribed in Branchini et al. Anal. Biochem. 396(2010): 290-297). Insome embodiments, the synthetic biomarker may be a contrast agent, anenzyme producing a detectable molecule, or a transporter drivingaccumulation of a detectable molecule. The synthetic biomarker may bemeasured in situ within subject's body. The synthetic biomarker may beselected from the group consisting of a photoacoustic reporter, abioluminescent reporter, an autofluorescent reporter, a chemiluminescentreporter, a luminescent reporter, or a colorimetric reporter, or anycombination thereof.

In some cases, at least one of the plurality of polypeptides or nucleicacid biomarker sequences may encode polypeptides or nucleic acidsdetectable in a biological sample from the subject. When the biomarkeris a polypeptide, the polypeptide may comprise an N-terminal secretionsignal sequence (e.g. the N-terminal signal peptide from CD33 or CD8a).Exemplary polypeptide biomarkers include, but are not limited to,photoacoustic reporters, bioluminescent reporters, autofluorescentreporters, chemiluminescent reporters, luminescent reporters,colorimetric reporters, and any combination thereof. Autofluorescentreporters include GFP, mCherry, or derivatives thereof. Colorimetricreporters include pigment-producing enzymes such as β-galactosidase(e.g. in combination with administration of X-gal), and tyrosinase.Bioluminescent, chemiluminescent or luminescent reporters includeluciferases (e.g. in combination with administration of coelenterazinesdescribed herein), including Gaussia luciferases, Renilla luciferases,and Photinus luciferases (e.g. including the engineered Ppy RE8 and RE9versions described in Branchini et al. Anal. Biochem. 396(2010):290-297). Reporters detectable by photoacoustic imaging include thepigment-producing enzymes such as β-galactosidase (e.g. in combinationwith administration of X-gal) and tyrosinase, autofluorescent proteins(e.g. GFP, mCherry, or derivatives thereof), non-fluorescent GFP-likechromoproteins (e.g. aeCP597 and cjBlue and derivatives thereof),bacteriophytochrome-based near-infrared fluorescent proteins (e.g.IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713/V256C,iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphP1-FP/C20S, orAphB variants), and reversibly photoswitchable proteins (e.g. Dronpa,Dronpa-M159T, and BphP1 or variants thereof). The synthetic biomarkermay be measured in situ within subject's body. The synthetic biomarkermay be selected from the group consisting of a photoacoustic reporter, abioluminescent reporter, an autofluorescent reporter, a chemiluminescentreporter, a luminescent reporter, or a colorimetric reporter, or anycombination thereof.

In some cases, the nucleic acid biomarker is e.g. a natural orengineered miRNA, an RNA hairpin, RNA aptamers or barcoded versionsthereof. The detectable nucleic acid biomarker may be a ribozyme, aself-splicing intron, an RNA hairpin, a microRNA, or barcoded versionsthereof, or other types of quantifiable RNA. The quantifiable nucleicacid may comprise a unique sequence detectable by quantitative PCR orhybridization-based techniques. When the nucleic acid is an miRNA, themiRNA may be detected e.g. by standard library generation techniquessuch as degenerate primer-based annealing and ligation, poly(A)polymerase labeling followed by RT or ligation, or sequential adapterligation coupled to q-PCR, sequencing, or an electrophoretic detectionmethod. When the biomarker is a polypeptide, the polypeptide maycomprise an N-terminal secretion signal sequence (e.g. the N-terminalsignal peptide from CD33 or CD8a).

When the nucleic acid is an engineered miRNA, the nucleic acid may bethe Sec-miR or miR-neg constructs described in Ronald et al. (Ronald etal. PLoS ONE 11(7): e0159369.) Such constructs comprise: (a) a codingsequence not expressed endogenously and not having any known vertebratetarget (e.g. Sec-miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) miR backbonesequences providing processing of pre-miRNA to mature miRNA flanking thecoding sequence (e.g. miR-155 or miR-130 backbone sequences); and (c) anEXOmotif enhancing loading into exosomes (e.g. GGAG). Such miRNAconstructs may be expressed in e.g. the 3′-UTR of a gene encoding areporter polypeptide, or from the 3′-UTR of a gene encoding a suitablynon-toxic protein (e.g. an endogenous structural protein such as actinor tubulin, or a highly expressed protein such as ubiquitin). In someembodiments, multiple copies (e.g. at least 2, at least 4) of theengineered miRNA may be provided in tandem.

In certain embodiments, the barcode molecule may uniquely identify adisease-specific promoter of said genetic construct. The barcodemolecule may comprise a nucleotide sequence or a peptide sequence. Incertain embodiments, the barcode molecule may comprise a unique DNA orRNA. When the barcode molecule comprises RNA, the RNA is a barcodeprocessed from a miRNA scaffold. In certain embodiments, the miRNAscaffold comprises 5′, 3′, and loop regions derived said miRNA scaffold,and stem regions comprising said barcode. In certain embodiments, themiRNA may be an engineered miRNA as described herein. When the barcodecomprises a peptide sequence, the barcode comprises an enzyme reporter.The peptide sequences may comprise an N-terminal secretion signalsequence as described herein. Further, the peptide sequences may bedetectable by non-invasive imaging as described herein.

In some cases, the at least one vector may be any of the vectorsdescribed herein. In some embodiments, the genetic construct comprises anon-viral vector. In some embodiments, the non-viral vector is ananoplasmid. In some embodiments, the genetic construct comprises areplication-incompetent recombinant virion or an isolated invertedterminal repeat (ITRs) derived therefrom. In some embodiments, thevirion may be a lentiviral, adeno-associated viral, adenoviral, orgamma-retroviral virion. In some embodiments, the virion is derived froma virus with primarily episomal genome maintenance within infectedcells. In some embodiments, the replication-incompetent virion is arecombinant adenovirus vector. In some embodiments, the AAV is serotype1, 2, 3, 4, 5, 6, 8, 9, Ad5, Ad-RGD, or Ad-19a/64, or a pseudotypedvariant thereof.

In some cases, the plurality of different promoters operably linked to aplurality of different nucleic acid sequences may comprise at least onepromoter. The plurality of different promoters operably linked to aplurality of different nucleic acid sequences may comprise at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15 promoters. The plurality of different promotersoperably linked to a plurality of different nucleic acid sequences maycomprise at most 2, at most 3, at most 4, at most 5, at most 6, at most7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13,at most 14, or at most 15 promoters.

In some cases, the plurality of different promoters operably linked to aplurality of different nucleic acid sequences may comprise at least onepromoter from Table 2. The plurality of different promoters operablylinked to a plurality of different nucleic acid sequences may compriseat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, or at least 15 promoters from Table 2. The plurality ofdifferent promoters operably linked to a plurality of different nucleicacid sequences may comprise at most 2, at most 3, at most 4, at most 5,at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, atmost 12, at most 13, at most 14, or at most 15 promoters from Table 2.

In some aspects, the present disclosure provides for a method fordetecting a disease in a subject, comprising: (a) administering to asubject a composition comprising the at least one vector above (e.g.comprising a plurality of different promoters operably linked to aplurality of different nucleic acid sequences, wherein the promotersdrive expression of the plurality of nucleic acid sequences in a cell toyield a plurality of polypeptides or nucleic acid biomarker sequences,wherein the promoters induce expression of the plurality of polypeptidesor nucleic acid biomarker sequences in a diseased cell preferentiallyover expression of the plurality of polypeptides or nucleic acidbiomarker sequences in non-diseased cells in a subject such that arelative ratio of the plurality of polypeptides or nucleic acidbiomarker sequences expressed in the diseased cell over the non-diseasedcells is greater than 1.0); (b) detecting said plurality of polypeptidesor nucleic acid biomarker sequences to obtain an expression profile; and(c) detecting said diseased cell based said expression profile, therebydetecting said disease.

In some cases, the method for detecting the disease in the subjectcomprises detecting said plurality of polypeptides or nucleic acidbiomarker sequences from a sample from the subject. In some cases, themethod may comprise isolating a biological sample from the subject. Thebiological sample may be a sample collected by a non-invasive methodfrom the subject. Exemplary non-invasive samples include, but are notlimited to, samples comprised of naturally shed bodily substances ornon-destructive scraping of externally accessible tissues, such assaliva, sputum, sweat, urine, stool, semen, mucus, cervicovaginalsecretions, breast milk, rheum, tears, and cheek epithelial swabs. Thebiological sample may be a sample collected by a minimally-invasivemethod from the subject. Exemplary minimally-invasive samples include,but are not limited to, blood samples or fractions thereof (e.g.obtained by venipuncture or capillary tube), pleural fluid samples (e.g.obtained by thoracentesis), amniotic fluid samples (e.g. obtained byamniocentesis), and gastric fluid samples (e.g. obtained by gastriclavage). The biological sample may be a sample obtained by biopsy, suchas a skin biopsy sample (e.g. obtained by punch, shave, saucerization,wedge, incisional, or excisional biopsy), a bone marrow sample (e.g.obtained by aspiration biopsy), a lymph node or breast biopsy (e.g.obtained by fine-needle aspiration, core needle biopsy, vacuum assistedbiopsy, or image-guided biopsy), a surgical biopsy sample (e.g. of aninternal organ obtained by excisional or incisional biopsy), or a mouth,GI-tract, lung, bladder, or urinary tract biopsy (e.g. obtained byendoscopy).

In some cases, the biological sample may be obtained a certain period oftime after administration of the composition comprising the at least onevector. The biological sample may be obtained at least about 15 minutes,at least about 30 minutes, at least about 1 hour, at least about 2hours, at least about 4 hours, at least about 8 hours, at least about 16hours, at least about 24 hours, at least about 36 hours, at least about48 hours, at least about 3 days, at least about 4 days, at least about 5days, at least about 6 days, at least about 7 days, at least about 8days, at least about 9 days, at least about 10 days, at least about 11days, at least about 12 days, at least about 13 days, at least about 14days, at least about 15 days, at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, or at least about 6 months after administration of thecomposition comprising the at least one vector. The biological samplemay be obtained at most about 15 minutes, at most about 30 minutes, atmost about 1 hour, at most about 2 hours, at most about 4 hours, at mostabout 8 hours, at most about 16 hours, at most about 24 hours, at mostabout 36 hours, at most about 48 hours, at most about 3 days, at mostabout 4 days, at most about 5 days, at most about 6 days, at most about7 days, at most about 8 days, at most about 9 days, at most about 10days, at most about 11 days, at most about 12 days, at most about 13days, at most about 14 days, at most about 15 days, at most about 1month, at most about 2 months, at most about 3 months, at most about 4months, at most about 5 months, or at most about 6 months afteradministration of the composition comprising the at least one vector. Insome embodiments, the biological sample may be obtained, and anybiomarker detection protocols performed multiple times afteradministration of the composition comprising the at least one vector(e.g. to monitor synthetic biomarker levels over time). The biologicalsample may be obtained at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, or 25 times after administration of the composition comprising theat least one vector. The biological sample may be obtained weekly ormonthly after administration of the composition comprising the at leastone vector.

In some cases, the composition comprising the at least one vector may beadministered intravenously, subcutaneously, intraventricularly,intrathecally, intracerebroventricularly, transdermally,intramuscularly, orally, by inhalation, nasally, rectally,intratumorally, or proxi-tumorally to the subject. Proxi-tumorally maydenote administration to the tissue within proximity of a tumor, oradministration into a region that would be predicted to be accessible tothe tumor via the lymphatic system (e.g. an adjoining lymph node).Intratumoral or proxi-tumoral approaches may involve the use ofadditional imaging techniques such as e.g. endoscopic ultrasonography(see e.g. Shirley et al. Gastroenterol Res Pract. 2013; 2013: 207129) orvia a brochioscope (see e.g. Rojas-Solano et al. J Bronchology InteryPulmonol. 2018 July; 25(3): 168-175). In some embodiments, thecomposition comprising the at least one vector may be administered intoat least one of the cervical, epitrochlear, supraclavicular, cervical,axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, orpopliteal lymph nodes. In some cases, lymph-node based administrationmay serve as a method of centralized local delivery to a tissue region.

In some cases, the composition comprising the at least one vector maycomprise a transfection agent as described herein.

In some cases, the composition comprising the at least one vector maycomprise a pharmaceutically acceptable carrier, as described herein.

In some cases, the method for detecting the disease in the subject maycomprise applying a machine learning or classifier algorithm to saidexpression profile, wherein the machine learning or classifier algorithmis configured to distinguish between an expression profile indicative ofa diseased cell from an expression profile indicative of a non-diseasedcell.

Machine learning or classifier algorithms refer generally to supervisedlearning and classification approaches executed by a computer system. Insupervised learning approaches, a group of samples from two or moregroups (e.g. diseased and non-diseased) are analyzed with a statisticalclassification method. Biomarker presence/absence/level data can be usedas a classifier that differentiates between the two or more groups. Anew sample can then be analyzed so that the classifier can associate thenew sample with one of the two or more groups. Commonly used supervisedclassifiers/classifier algorithms include without limitation the neuralnetwork (multi-layer perceptron), support vector machines, k-nearestneighbors, Gaussian mixture model, Gaussian, naive Bayes, decision treeand radial basis function (RBF) classifiers. Linear classificationmethods include Fisher's linear discriminant, logistic regression, naiveBayes classifier, perceptron, and support vector machines (SVMs). Otherclassifiers/classifier algorithms for use with the invention includequadratic classifiers, k-nearest neighbor, boosting, decision trees,random forests, neural networks, pattern recognition, Bayesian networksand Hidden Markov models.

Classification using supervised methods is generally performed by thefollowing methodology:

In order to solve a given problem of supervised learning (e.g. learningto recognize handwriting) one has to consider various steps:

1. Gather a training set. These can include, for example, samples thatare from a food or environment contaminated or not contaminated with aparticular microbe, samples that are contaminated with differentserotypes of the same microbe, samples that are or are not contaminatedwith a combination of different species and serotypes of microbes, etc.The training samples are used to “train” the classifier.

2. Determine the input “feature” representation of the learned function.The accuracy of the learned function depends on how the input object isrepresented. Typically, the input object is transformed into a featurevector, which contains a number of features that are descriptive of theobject. The number of features should not be too large, because of thecurse of dimensionality; but should be large enough to accuratelypredict the output. The features might include a set of bacterialspecies or serotypes present in a food or environmental sample derivedas described herein.

3. Determine the structure of the learned function and correspondinglearning algorithm. A learning algorithm is chosen, e.g., artificialneural networks, decision trees, Bayes classifiers or support vectormachines. The learning algorithm is used to build the classifier.

4. Build the classifier (e.g. classification model). The learningalgorithm is run on the gathered training set. Parameters of thelearning algorithm may be adjusted by optimizing performance on a subset(called a validation set) of the training set, or via cross-validation.After parameter adjustment and learning, the performance of thealgorithm may be measured on a test set of naive samples that isseparate from the training set.

Once the machine learning classifier (e.g. classification model) isdetermined as described above, it can be used to classify a sample,e.g., a sample from a subject that has received the compositioncomprising the at least one vector above.

In some cases, the relative pattern of biomarker or barcode moleculeexpression from the plurality of vectors provides a “unique fingerprint”corresponding to one or more different types of cancers

In some cases, a method by which artificial intelligence and machinelearning can be applied to the patterns of biomarker expression in orderto develop a predictive series of plurality of vectors for accuratecancer detection.

In some cases, the method comprises detecting the polypeptide or nucleicacid sequence encoded by the at least one vector. The detecting may alsocomprise an immunoassay. Immunoassays include those described in e.g.,U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124;5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526;5,525,524; and 5,480,792. Immunoassays include various sandwich,competitive, or non-competitive assay formats, which generate a signalthat is related to the presence or amount of a protein analyte ofinterest. Any suitable immunoassay may be utilized, for example, lateralflow, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs),competitive binding assays, and the like. When the polypeptide is areporter polypeptide, the detecting may comprise a photoacoustic assay,a bioluminescence assay, a fluorescence assay, a chemiluminescent assay,a colorimetric assay, or any combination thereof.

The method of detection may comprise sequencing. Sequencing methods mayinclude: Next Generation sequencing, high-throughput sequencing,pyrosequencing, classic Sanger sequencing methods,sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (LifeTechnologies/Thermo-Fisher), massively-parallel sequencing, clonalsingle molecule Array (Solexa), shotgun sequencing, Maxim-Gilbertsequencing, and primer walking.

The detection may comprise a “real time amplification” method also knownas quantitative PCR (qPCR) or Taqman (see, e.g., U.S. Pat. No. 5,210,015to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No.5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research,6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001(1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280,(1991); and Livak, K. J., et al., PCR Methods and Applications 357-362(1995)). The basis for this method of monitoring the formation ofamplification product is to measure continuously PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe. Theprobe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye. The probe is designed tohave at least substantial sequence complementarity with a site on thetarget mRNA or nucleic acid derived from. Upstream and downstream PCRprimers that bind to flanking regions of the locus are also added to thereaction mixture. When the probe is intact, energy transfer between thetwo fluorophores occurs and the quencher quenches emission from thereporter. During the extension phase of PCR, the probe is cleaved by the5′ nuclease activity of a nucleic acid polymerase such as Taqpolymerase, thereby releasing the reporter from thepolynucleotide-quencher and resulting in an increase of reporteremission intensity which can be measured by an appropriate detector. Therecorded values can then be used to calculate the increase in normalizedreporter emission intensity on a continuous basis and ultimatelyquantify the amount of the mRNA being amplified.

In some embodiments, for qPCR or Taqman detection, an RT-PCR step may befirst performed to generate cDNA from cellular RNA. Such amplificationby RT-PCR can either be general (e.g. amplification with partially/fullydegenerate oligonucleotide primers) or targeted (e.g. amplification witholigonucleotide primers directed against specific genes which are to beanalyzed at a later step).

In some embodiments, qPCR or Taqman may be used immediately following areverse-transcriptase reaction performed on isolated cellular mRNA; thisvariety serves to quantitate the levels of individual mRNAs during qPCR.

In some embodiments, for qPCR or Taqman detection or RNA sequencing, a“pre-amplification” step may be first performed on cDNA transcribed fromcellular RNA. This serves to increase signal in conditions where thenatural level of the RNA/cDNA to be detected is very low. Suitablemethods for pre-amplification include but are not limited LM-PCR, PCRwith random oligonucleotide primers (e.g. random hexamer PCR), PCR withpoly-A specific primers, and any combination thereof. Thepre-amplification may be either general or targeted in the same way asthe reverse-transcription reaction described above.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentdisclosure to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

TABLE 1 Example Sequences Useful with Methods and CompositionsAccording to the Disclosure SEQ ID NO: NAME SEQUENCE 1 MC-pSurv-SEAP-CCCCAACTGGGGTAACCTTTGGGCTCCCCGGGCGCGACTAGT WPRE-pAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTT Mini circleTTGTGTGAATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTACTAGTGCCATAGAACCAGAGAAGTGAGTGGATGTGATGCCCAGCTCCAGAAGTGACTCCAGAACACCCTGTTCCAAAGCAGAGGACACACTGATTTTTTTTTTAATAGGCTGCAGGACTTACTGTTGGTGGGACGCCCTGCTTTGCGAAGGGAAAGGAGGAGTTTGCCCTGAGCACAGGCCCCCACCCTCCACTGGGCTTTCCCCAGCTCCCTTGTCTTCTTATCACGGTAGTGGCCCAGTCCCTGGCCCCTGACTCCAGAAGGTGGCCCTCCTGGAAACCCAGGTCGTGCAGTCAACGATGTACTCGCCGGGACAGCGATGTCTGCTGCACTCCATCCCTCCCCTGTTCATTTGTCCTTCATGCCCGTCTGGAGTAGATGCTTTTTGCAGAGGTGGCACCCTGTAAAGCTCTCCTGTCTGACTTTTTTTTTTTTTTTAGACTGAGTTTTGCTCTTGTTGCCTAGGCTGGAGTGCAATGGCACAATCTCAGCTCACTGCACCCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGTTGGGATTACAGGCATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAAGGTTTCACCGTGATGGCCAGGCTGGTCTTGAACTCCAGGACTCAAGTGATGCTCCTGCCTAGGCCTCTCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACCCGGCCTGCACGCGTTCTTTGAAAGCAGTCGAGGGGGCGCTAGGTGTGGGCAGGGACGAGCTGGCGCGGCGTCGCTGGGTGCACCGCGACCACGGGCAGAGCCACGCGGCGGGAGGACTACAACTCCCGGCACACCCCGCGCCGCCCCGCCTCTACTCCCAGAAGGCCGCGGGGGGTGGACCGCCTAAGAGGGCGTGCGCTCCCGACATGCCCCGCGGCGCGCCATTAACCGCCAGATTTGAATCGCGGGACCCGTTGGCAGAGGTGGGAATTCACCGGTCACCATGGTTCTGGGGCCCTGCATGCTGCTGCTGCTGCTGCTGCTGGGCCTGAGGCTACAGCTCTCCCTGGGCATCATCCCAGTTGAGGAGGAGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTGCAGCCTGCACAGACAGCCGCCAAGAACCTCATCATCTTCCTGGGCGATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCCTAAAAGGGCAGAAGAAGGACAAACTGGGGCCTGAGATACCCCTGGCTATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATACAATGTAGACAAACATGTGCCAGACAGTGGAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGCTTGAGTGCAGCCGCCCGCTTTAACCAGTGCAACACGACACGCGGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCAGGGAAGTCAGTGGGAGTGGTAACCACCACACGAGTGCAGCACGCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAACTGGTACTCGGACGCCGACGTGCCTGCCTCGGCCCGCCAGGAGGGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGACATTGATGTGATCCTGGGTGGAGGCCGAAAGTACATGTTTCGCATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTGGGACCAGGCTGGACGGGAAGAATCTGGTGCAGGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTATGTGTGGAACCGCACTGAGCTCATGCAGGCTTCCCTGGACCCGTCTGTGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGACTCCACACTGGACCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCGTGGAGGGTGGTCGCATCGACCACGGTCATCACGAAAGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCATTGAGAGGGCGGGCCAGCTCACCAGCGAGGAGGACACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGCTACCCCCTGCGAGGGAGCTCCATCTTCGGGCTGGCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGAAACGGTCCAGGCTATGTGCTCAAGGACGGCGCCCGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAGTCAGCAGTGCCCCTGGACGAAGAGACCCACGCAGGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCACGGCGTGCAGGAGCAGACCTTCATAGCGCACGTCATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCCCCCGCCGGCACCACCGACGCCGCGCACCCGGGGCGGTCCCGGTCCAAGCGTCTGGATTGAGCTAGCTTCGAATTTAAATCGGATCCCTGCAGGAGCTCGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAAATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCTCCCGCCCCTAACTCCGCCCAATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGACTTT TGCAGATCGACCCATGGGGGCCCG 2MC-pSurv-Luc2- CCCCAACTGGGGTAACCTTTGGGCTCCCCGGGCGCGACTAGT WPRE-pAAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTACTAGTGAATTGATGCCATAGAACCAGAGAAGTGAGTGGATGTGATGCCCAGCTCCAGAAGTGACTCCAGAACACCCTGTTCCAAAGCAGAGGACACACTGATTTTTTTTTTAATAGGCTGCAGGACTTACTGTTGGTGGGACGCCCTGCTTTGCGAAGGGAAAGGAGGAGTTTGCCCTGAGCACAGGCCCCCACCCTCCACTGGGCTTTCCCCAGCTCCCTTGTCTTCTTATCACGGTAGTGGCCCAGTCCCTGGCCCCTGACTCCAGAAGGTGGCCCTCCTGGAAACCCAGGTCGTGCAGTCAACGATGTACTCGCCGGGACAGCGATGTCTGCTGCACTCCATCCCTCCCCTGTTCATTTGTCCTTCATGCCCGTCTGGAGTAGATGCTTTTTGCAGAGGTGGCACCCTGTAAAGCTCTCCTGTCTGACTTTTTTTTTTTTTTTAGACTGAGTTTTGCTCTTGTTGCCTAGGCTGGAGTGCAATGGCACAATCTCAGCTCACTGCACCCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGTTGGGATTACAGGCATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAAGGTTTCACCGTGATGGCCAGGCTGGTCTTGAACTCCAGGACTCAAGTGATGCTCCTGCCTAGGCCTCTCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACCCGGCCTGCACGCGTTCTTTGAAAGCAGTCGAGGGGGCGCTAGGTGTGGGCAGGGACGAGCTGGCGCGGCGTCGCTGGGTGCACCGCGACCACGGGCAGAGCCACGCGGCGGGAGGACTACAACTCCCGGCACACCCCGCGCCGCCCCGCCTCTACTCCCAGAAGGCCGCGGGGGGTGGACCGCCTAAGAGGGCGTGCGCTCCCGACATGCCCCGCGGCGCGCCATTAACCGCCAGATTTGAATCGCGGGACCCGTTGGCAGAGGTGGAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAATCTAGAGCTAGCGAATTCAGATCTGATATCTCTAGAGTCGAGCTAGCTTCGAATTTAAATCGGATCCCTGCAGGAGCTCGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAAATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCTCCCGCCCCTAACTCCGCCCAATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGACTTTTGCAGATCGA CCCATGGGGGCCCG 3Mini R6K origin GGCTTGTTGTCCACAACCGTTAAACCTTAAAAGCTTTAAAAG (mini-ori)#1CCTTATATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTTAGCCATGAGAGCTTAGTACGTTAGCCATGAGGGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTAAACATGAGAGCTTAGTA CGTACTATCAACAGGTTGAACTGCTGATC4 RNA-OUT GTAGAATTGGTAAAGAGAGTCGTGTAAAATATCGAGTTCGCA SELECTABLECATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGA MARKER #1TCATATGACAAGATGTGTATCTACCTTAACTTAATGATTTTG ATAAAAATCATTA 5 ExampleGCCATAGAACCAGAGAAGTGAGTGGATGTGATGCCCAGCTCC nanoplasmid withAGAAGTGACTCCAGAACACCCTGTTCCAAAGCAGAGGACACA SEAP insert,CTGATTTTTTTTTTTAATAGGCTGCAGGACTTACTGTTGGTG WPRE element,GGACGCCCTGCTTTGCGAAGGGAAAGGAGGAGTTTGCCCTGA survivin promoter,GCACAGGCCCCCACCCTCCACTGGGCTTTCCCCAGCTCCCTT R6K origin, RNA-GTCTTCTTATCACGGTAGTGGCCCAGTCCCTGGCCCCTGACT out selectableCCAGAAGGTGGCCCTCCTGGAAACCCAGGTCGTGCAGTCAAC markerGATGTACTCGCCGGGACAGCGATGTCTGCTGCACTCCATCCCTCCCCTGTTCATTTGTCCTTCATGCCCGTCTGGAGTAGATGCTTTTTGCAGAGGTGGCACCCTGTAAAGCTCTCCTGTCTGACTTTTTTTTTTTTTTTAGACTGAGTTTTGCTCTTGTTGCCTAGGCTGGAGTGCAATGGCACAATCTCAGCTCACTGCACCCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGTTGGGATTACAGGCATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAAGGTTTCACCGTGATGGCCAGGCTGGTCTTGAACTCCAGGACTCAAGTGATGCTCCTGCCTAGGCCTCTCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACCCGGCCTGCACGCGTTCTTTGAAAGCAGTCGAGGGGGCGCTAGGTGTGGGCAGGGACGAGCTGGCGCGGCGTCGCTGGGTGCACCGCGACCACGGGCAGAGCCACGCGGCGGGAGGACTACAACTCCCGGCACACCCCGCGCCGCCCCGCCTCTACTCCCAGAAGGCCGCGGGGGGTGGACCGCCTAAGAGGGCGTGCGCTCCCGACATGCCCCGCGGCGCGCCATTAACCGCCAGATTTGAGTCGCGGGACCCGTTGGCAGAGGTGGGAATTCACCGGTCACCATGGTTCTGGGGCCCTGCATGCTGCTGCTGCTGCTGCTGCTGGGCCTGAGGCTACAGCTCTCCCTGGGCATCATCCCAGTTGAGGAAGAGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTGCAGCCTGCACAGACAGCCGCCAAGAACCTCATCATCTTCCTGGGCGATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCCTAAAAGGGCAGAAGAAGGACAAACTGGGGCCTGAGATACCCCTGGCTATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATACAATGTAGACAAACATGTGCCAGACAGTGGAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGCTTGAGTGCAGCCGCCCGCTTTAACCAGTGCAACACGACACGCGGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCAGGGAAGTCAGTGGGAGTGGTAACCACCACACGAGTGCAGCACGCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAACTGGTACTCGGACGCCGACGTGCCTGCCTCGGCCCGCCAGGAGGGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGACATTGATGTGATCCTGGGTGGAGGCCGAAAGTACATGTTTCGCATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTGGGACCAGGCTGGACGGGAAGAATCTGGTGCAGGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTATGTGTGGAACCGCACTGAGCTCATGCAGGCTTCCCTGGACCCGTCTGTGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGACTCCACACTGGACCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCGTGGAGGGTGGTCGCATCGACCACGGTCATCACGAAAGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCATTGAGAGGGCGGGCCAGCTCACCAGCGAGGAGGACACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGCTACCCCCTGCGAGGGAGCTCCATCTTCGGGCTGGCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGAAACGGTCCAGGCTATGTGCTCAAGGACGGCGCCCGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAGTCAGCAGTGCCCCTGGACGAAGAGACCCACGCAGGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCACGGCGTGCAGGAGCAGACCTTCATAGCGCACGTCATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCCCCCGCCGGCACCACCGACGCCGCGCACCCGGGGCGGTCCCGGTCCAAGCGTCTGGATTGAGCTAGCTTCGAATTTAAATCGGATCCCTGCAGGAGCTCGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTACAAGTAACCGCGAATTCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCCCGGGACGGCCGCTAGCCCGCCTAATGAGCGGGCTTTTTTTTGGCTTGTTGTCCACAACCGTTAAACCTTAAAAGCTTTAAAAGCCTTATATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTTAGCCATGAGAGCTTAGTACGTTAGCCATGAGGGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTAAACATGAGAGCTTAGTACGTACTATCAACAGGTTGAACTGCTGATCCACGTTGTGGTAGAATTGGTAAAGAGAGTCGTGTAAAATATCGAGTTCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATCATATGACAAGATGTGTATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGGTACGGCCGCGGTGCCAGGGCGTGCCCTTGGGCTCCC CGGGCGCGACTAGT

EXAMPLES Example 1.—Plasmid and Minicircle Construction

All plasmids were constructed using standard PCR and cloning technologyand sequenced by Sequetech (Mountain View, Calif.). To generate bothparental plasmids (PP) and MCs the system described by Kay et al.,(2010) Nat. Biotechnol. 28: 1287-1289, incorporated herein by referencein its entirety was used (System Biosciences, Mountain View, Calif.).The 977 base-pair (bp) Survivin promoter was sub-cloned from pSurv-FL(Ray S, et al. (2008) Molecular therapy: J. Am. Soc. Gene Therapy16:1848-1856) into the MN-100 PP backbone (System Biosciences, MountainView, Calif.) containing a SV40 polyA and Woodchuck Hepatitis virusposttranscriptional element (WPRE) to generate PP-pSurv-WPRE. Next, theSEAP transgene from pSELECT-zeo-SEAP (Invivogen, San Diego, Calif.) wassubcloned into PP-pSurv-WPRE to generate PP-pSurv-SEAP-WPRE (FIG.2A-top). Both PP-pSurv-SEAP-WPRE (PP) and MC-pSurv-SEAP-WPRE (MC) (FIG.2A-bottom) were amplified and purified according to the protocoloutlined in Kay et al., (2010) Nat. Biotechnol. 28: 1287-1289) and thesupplier's instructions (System Biosciences, Mountain View, Calif.).

ZYCY10P3S2T E. coli were transformed with the PP, colonies were picked,and E. coli were grown overnight in TB broth. To generate MCs,site-specific recombination via expression of phiC31 integrase wasinitiated by addition of equal volume of LB broth containing 0.001%L-arabinose and 16 mL NaOH, and cultures were grown for an additional5.5 h at 30° C. For the PP, the cells were grown in the same mediawithout L-arabinose supplementation. Endotoxin-free mega kits (Qiagen,Valencia, Calif.) were used to purify both PP and MC.

Example 2.—Cell Culture and Transfection

MDA-MB-231 (ATCC, Manassas, Va.), MeWo (ATCC, Manassas, Va.) andSK-MEL-28 human melanoma cell lines were maintained on MEM and DMEM(Gibco, Carlsbad, Calif.), respectively. Media was supplemented with 10%Fetal Bovine Serum (FBS) and 1× Antibiotic-Antimycotic solution (LifeTechnologies) and cells were maintained in 5% CO2 incubator at 37° C.

Cell lines were plated (1.25×10⁵ cells per well) in 24-well plates 1 dayprior to transfection. Cells were transfected with equal mass of PP orMC (1 μg) and 2 μl of a linear polyethylenimine transfection agent(jetPEI, Polyplus transfection, Illkirch, France) according to themanufacturer's instructions. Medium was collected daily, centrifuged at1200 rpm for 3 minutes and the supernatant was stored at −20° C. untilSEAP concentrations were measured. Following medium collection, eachwell was washed with PBS and fresh medium was added; therefore, SEAPmeasurements reflect protein production over a 24-hour period.

Example 3.—Subcutaneous Tumor Model and Intratumoral Administration ofMinicircles

2×10⁶ MeWo cells were implanted into the right flank of female nude mice(Nu/Nu; Charles River) and tumors developed over a period of 3 weeks(n=4). MCs (20 μg) were complexed with a linear polyethyleniminetransfection agent (in vivo-jetPEI, Polyplus transfection, Illkirch,France) at an N/P ratio of 6 (N/P is the number of nitrogen residues inin vivo jetPEI per nucleic acid phosphate) and resuspended in 50 μL 5%glucose.

Intratumoral (I.T.) injections were performed over approximately 2 minby injecting DNA-transfection agent complexes at multiple loci withineach tumor. Two cohorts of control mice received either an intramuscular(I.M.) injection of MC at the same dose (n=3) or an I.T. injection of 5%glucose only (n=3).

Example 4.—Experimental Melanoma Metastases Model, BLI, and SystemicAdministration of Minicircles

To evaluate the ability to detect tumors after systemic administrationof MCs, an experimental metastases model described previously (Bhang etal., (2011) Nat. Med. 17:123-129) was used. 5×10⁶ MeWo cells stablyexpressing a BRET fusion protein (RLuc8.6-TurboFP-BRET6)(Dra-gulescu-Andrasi et al., (2011) Proc. Nat. Acad. Sci. U.S.A.108:12060-12065) were injected into irradiated (5 Gy) female nude mice(Nu/Nu; Charles River) via the tail-vein (200 μL of PBS total volume).At weekly intervals following cell injection, tumor development wasmonitored with BLI immediately following intravenous administration ofthe substrate coelenterazine (35 μg/mouse; diluted in 150 μl of PBS)using an IVIS-200 imaging system (PerkinElmer). Using the softwarepackage Living Image 4.1, region of interests (ROIs) were drawn over thelungs in each image to quantitate tumor burden. BLI data is expressed aslung average radiance in photons/second/cm2/steradian.

Tumor-bearing mice (n=7) or irradiated control mice (n=7) wereadministered 40 μg of MC complexed with a linear polyethyleniminetransfection agent (N/P ratio of 8; in vivo-jetPEI, Polyplustransfection, Illkirch, France) and resuspended in 400 μl of 5% glucose.Mice were then injected via the tail-vein with two 200 μl injections anda gap of 5 minutes between the first and second injection. An additionalcontrol group (n=5) of irradiated mice were administered 400 μl of 5%glucose alone.

Example 5.—Plasma Collection

Blood samples were collected via the submandibular vein at least 1 dayprior to MC injection and for up to 2 weeks following injection. Blood(approximately 75-100 ml) was collected in lithium heparin-coatedmicrotubes (BD), kept on ice before processing, and then centrifuged at10,000×g for 5 minutes at 4° C. Plasma was collected and stored at −80°C. prior to SEAP measurements.

Example 6.—SEAP Assay

To measure SEAP concentration in both medium and plasma the Great EscAPeSEAP Chemiluminescence Assay kit 2.0 according the manufacturer'sinstructions (Clontech) was used. Briefly, 25 μl of medium or plasma wasadded to 1× dilution buffer, and endogenous alkaline phosphatase washeat-inactivated at 65° C. for 30 minutes. Samples were put on ice for 3minutes and then allowed to recover to room temperature. 100 μl of SEAPsubstrate was added, incubated for 30 minutes at room temperature, andluminescence (relative light units; RLU) was measured over 10 secondsusing a TD 20/20 luminometer (Turner Designs, Sunnyvale, Calif.).

Example 7.—Tumor-Activatable Minicircles are Advantageous Over PlasmidsAcross Multiple Melanoma Cell Lines

The transcriptional activity of two tumor-specific promoters, pSury andthe progression elevated gene-3 promoter (pPEG) (Bhang et al., (2011)Nat. Med. 17: 123-129) were compared to assess which promoter would givethe lowest background in healthy tissues. Plasmids expressing acodon-optimized firefly luciferase (Luc2) driven by either pSury or pPEGwere constructed and delivered systemically into healthy female Nu/Numice. After two days, pSurv-driven plasmids showed significantly lowerbackground Luc2 expression than pPEG driven constructs, particularly inthe heart and lung (FIGS. 18A-18D). The tumor-specific promoter activityin both primary human fibroblasts and two human tumor cell lines wasalso compared. Again, pSury had lower background activity in humanfibroblasts and equivalent or higher expression in tumor cell lines(FIGS. 19A-19C).

Tumor-activatable parental plasmids (PP; approximately 7.9 kb) andminicircles (MC; approximately 4.1 kb) with pSury driving SEAPexpression (FIGS. 2A-4) were developed. To compare SEAP concentrationattainable with these two constructs, two human melanoma cell lines(MeWo, FIG. 15; and SK-MEL-28, FIG. 20) were transfected with equal massof PP and MC and equal volume of a linear polyethylenimine (PEI)transfection agent. Equal mass was compared since the main advantage ofMC usage is their smaller size and the main dose limitation of non-viralDNA vectors in vivo is typically the amount of transfection agent, notthe amount of DNA. Following transfection, SEAP concentration wasmeasured in the culture medium each day for up to 8 days. Each datapoint reflected the SEAP accumulation within the previous 24-hour periodnot the cumulative SEAP concentration across multiple days. By day 3 inMeWo cells, MCs had significantly higher SEAP concentration in themedium compared to PPs and these differences were maintained until thelast day (day 8) of the experiment (FIG. 15). Similar results wereobtained for SK-MEL-28 cells. The only significant differences werenoted at day 2 (FIG. 20). Therefore, MCs driven by the tumor-activatablepSury have improved transgene expression profiles in melanoma cancercells compared to their parental plasmid counterparts. To ensure thatMCs provide an advantage over PPs in vivo, the transgene expressionlevels achieved by PPs and MCs driven by the strong constitutiveelongation factor-1 alpha promoter (pEF1) after systemic administrationin mice (n=5 for PP and n=4 for MC) were compared and foundsignificantly higher (p<0.05) lung expression with MCs at multiple timepoints post-delivery (FIGS. 21A and 21B).

Example 8.—Intratumoral Injection of Tumor-Activatable MCs Leads toDetectable Plasma SEAP Concentration

Since pSury transcriptional activity is relatively low compared tostrong promoters such as pCMV (FIGS. 18A-18D), it was determined whetherdirect intratumoral (I.T.) administration of tumor-activatable MCs wouldlead to a detectable SEAP signal in the blood. Mice bearing subcutaneousMeWo xenografts (approximately 50-80 mm³) were I.T. administered 20 μgof MCs complexed with PEI (n=4) or PBS only (n=3) and SEAP concentrationwas measured before and at 1, 3, 5, 7, 11 and 14 days after injection(FIG. 7). Standard curve analysis showed that SEAP measurements inplasma were reproducible over 5 orders of magnitude and a SEAPconcentration as low as 0.3 ng in 25 μl of plasma was detectable (FIGS.22A and 22B). By day 3, significantly (p<0.01) increased plasma SEAPconcentration was detected in mice receiving MC compared to control mice(FIGS. 15A-15D; p<0.01). Furthermore, significant differences betweenthese two groups were noted for up to 2 weeks post-administration. Thetumor specificity of expression was also examined by performingintramuscular (I.M.) MC injections on a group of mice (n=3). Nosignificant differences were noted between tumor-bearing mice receivingI.T. 5% glucose (Mock) injections or I.M. MC-injected mice. Hence, whenadequate transfection efficiency is achieved, pSurv-driventumor-activatable MCs produce SEAP within tumors at levels sufficientenough to be detectable in the blood at multiple time points followingadministration.

Example 9.—Systemic Injection of Tumor-Activatable MCs can IdentifyTumor-Bearing Subjects and Assess Tumor Burden

The ability of a measurement of plasma SEAP concentration followingsystemic administration of tumor-activatable MCs to distinguishtumor-bearing from healthy subjects was tested. MeWo melanoma cellsstably expressing a bioluminescence resonance energy transfer (BRET)fusion reporter were administered via the tail vein into irradiated nudemice (n=7) and tumor development was monitored over time withbioluminescence imaging (BLI) (FIGS. 15A-15C) and assessed qualitativelyat sacrifice (FIG. 23). Although a wide range of tumor burden wasobserved qualitatively 3 days prior to MC administration, all tumorswere primarily localized within the lungs (FIGS. 15A-15C). Followingsacrifice, as expected, multiple melanotic tumor foci were notedthroughout the lungs (FIG. 23). Based on changes in BLI signal, tumorswould have been approximately 4.5-fold smaller at the time of MCadministration (2 weeks prior).

For each mouse, plasma SEAP concentration was measured before (0 days)and at 1, 3, 7, 11 and 14 days after tail-vein administration of 40 μgof MC (Tumor+MC). As control groups, healthy (tumor-free) mice alsoreceived either MC (Control+MC; n=6) or 5% glucose (Control MC; n=5). Asseen in FIGS. 16A-16C, for individual tumor-bearing mice plasma SEAPconcentration was elevated post-MC injection. Regardless of tumorburden, the tumor-bearing mice showed significantly (p<0.05) higherplasma SEAP concentration profiles between days 3-14 post-administrationcompared to both control groups (FIG. 15). Some healthy mice receivingMC showed a slightly positive SEAP signal (most likely reflectingpromoter leakiness, as also noted with pSury in FIG. 18A-18D), overallno significant differences were noted between the two control micegroups (FIG. 16D). Therefore, measurement of plasma SEAP levelsfollowing systemic administration of tumor-activatable MCs coulddifferentiate between tumor-bearing and healthy subjects and a widewindow of opportunity (>1 week) was available to identify tumor-bearingsubjects.

Since SEAP levels were elevated at multiple time points following MCadministration, the cumulative shedding of SEAP into plasma wasevaluated by calculating the plasma SEAP concentration area under thecurve (AUC) for each mouse. Comparison of this single metric across allmice revealed no differences between the two control groups(Control+/−MC), but significantly (p<0.05) elevated values betweentumor-bearing mice and both control groups (FIG. 17A).

The ability of the assay to distinguish between tumor-bearing andhealthy subjects by performing receiver operator-characteristic curve(ROC) analysis was evaluated, as shown in FIG. 17B. This revealed asignificant (p<0.05) area of 0.918 (±0.084 SE) and a 95% confidenceinterval of 0.754 to 1.083. Hence, with this first-generation vectorused at the MC doses described, the assay was reliable in identifyingtumor-bearing subjects.

Some tumor-bearing subjects had AUC values that were only slightly abovethe mean of the control mice receiving MC FIG. 17A. Moreover, as shownin FIG. 16A-16C, the change of plasma SEAP concentration appeared toqualitatively correspond to the degree of tumor burden. Based on thesetwo observations, it was hypothesized that SEAP AUCs would correlatewith lung tumor burden (as assessed by BLI within 3 days prior to MCadministration). Since tumors were primarily located within the lungs,and the optical BLI signal is tissue-depth dependent, the evaluation wasrestricted to mice with only lung tumors (n=6). One mouse with multiplemetastatic foci outside the lung was excluded, although inclusion ofthis mouse showed an r² of 0.056 and a p-value of close to significance(p=0.0541). As expected, ROI analysis of the lung BLI signal prior to MCadministration revealed a wide range of lung tumor sizes (FIG. 17).Importantly, lung tumor burden was significantly correlated with SEAPAUC values (r²=0.714; p<0.05) (FIG. 16C). Therefore, ourtumor-activatable MC system not only shows a robust ability to identifytumor-bearing subjects but provided tumor burden is restricted to oneorgan can also be used to evaluate disease extent.

Example 10.—Statistical Analysis

All statistical analysis was performed using Prism 6.0 software(Graphpad software). Comparison of SEAP measurements from cell culturemedium was performed using two-way analysis of variance (ANOVA) followedby Sidak's multiple comparisons test. Longitudinal plasma SEAPmeasurements from mice were compared using two-way repeated measuresANOVA followed by Tukey's multiple comparisons test. Comparison of SEAPAUC measurements across mice cohorts was performed using a one-way ANOVAfollowed by Tukey's multiple comparisons test. ROC analysis wasperformed between SEAP AUC data from tumor-bearing and healthy micereceiving MC. Finally, Pearson correlation analysis of SEAP AUC and lungtumor burden measurements was performed. For all tests a nominal p-valueless than 0.05 was considered to be significant.

Example 11.—Discovery of New Promoters for Tumor Detection

Publicly-available databases were mined to discover new endogenous genes(and hence promoters) that may serve as part of synthetic biomarkerconstructs alternative to or in addition to Survivin/BIRC5. Two sourcesof data were utilized: tumor sample expression from The Cancer GenomeAtlas (TCGA), which has curated over 20,000 primary cancer samples from33 types of cancer against pair matched normal tissues; and theGenotype-Tissue Expression (GTEx) database, which contains diverseinformation on expression data in normal tissues. An algorithm wasapplied to the combined data set that ranked target genes by the numberof distinct tumor types in which the lowest quartile of that target'sgene expression was significantly higher than the highest quartile ofgene expression across all corresponding normal tissues (e.g. to findgenes that are expressed in several tumor types at levels above‘background’ level in all normal tissues). Normal tissue expression datafrom GTEx was selected as a better indicator of healthy normal tissuethan matching normal tissues from cancer patients in the TCGA database.The algorithm was run versus expression data from all 19,000 genes inthe human genome and the algorithm revealed overexpression of genesacross a substantial number of tumor types over all normal tissues. Manyof these genes are presented in Table 2 below.

TABLE 2 Genes for which tumor expression is significantly higher thanall normal tissues Gene Symbol Rank Name UBE2T 12 ubiquitin conjugatingenzyme E2 T [Source: HGNC Symbol; Acc: HGNC: 25009] CHEK1 10 checkpointkinase 1 [Source: HGNC Symbol; Acc: HGNC: 1925] ECT2 10 epithelial celltransforming 2 [Source: HGNC Symbol; Acc: HGNC: 3155] BCL2L12 9 BCL2like 12 [Source: HGNC Symbol; Acc: HGNC: 13787] CENPI 9 centromereprotein I [Source: HGNC Symbol; Acc: HGNC: 3968] E2F1 9 E2Ftranscription factor 1 [Source: HGNC Symbol; Acc: HGNC: 3113] FLAD1 9flavin adenine dinucleotide synthetase 1 [Source: HGNC Symbol; Acc:HGNC: 24671] PPM1G 9 protein phosphatase, Mg2+/Mn2+ dependent 1G[Source: HGNC Symbol; Acc: HGNC: 9278] UBE2S 9 ubiquitin conjugatingenzyme E2 S [Source: HGNC Symbol; Acc: HGNC: 17895] AUNIP 8 aurorakinase A and ninein interacting protein [Source: HGNC Symbol; Acc: HGNC:28363] CDC6 8 cell division cycle 6 [Source: HGNC Symbol; Acc: HGNC:1744] CENPL 8 centromere protein L [Source: HGNC Symbol; Acc: HGNC:17879] DNA2 8 DNA replication helicase/nuclease 2 [Source: HGNC Symbol;Acc: HGNC: 2939] DSN1 8 DSN1 homolog, MIS12 kinetochore complexcomponent [Source: HGNC Symbol; Acc: HGNC: 16165] DTYMK 8deoxythymidylate kinase [Source: HGNC Symbol; Acc: HGNC: 3061] GPRIN1 8G protein regulated inducer of neurite outgrowth 1 [Source: HGNC Symbol;Acc: HGNC: 24835] MTFR2 8 mitochondrial fission regulator 2 [Source:HGNC Symbol; Acc: HGNC: 21115] RAD51AP1 8 RAD51 associated protein 1[Source: HGNC Symbol; Acc: HGNC: 16956] SNRPA1 8 small nuclearribonucleoprotein polypeptide A′ [Source: HGNC Symbol; Acc: HGNC: 11152]ATAD2 7 ATPase family, AAA domain containing 2 [Source: HGNC Symbol;Acc: HGNC: 30123] BUB1 7 BUB1 mitotic checkpoint serine/threonine kinase[Source: HGNC Symbol; Acc: HGNC: 1148] CACYBP 7 calcyclin bindingprotein [Source: HGNC Symbol; Acc: HGNC: 30423] CDCA3 7 cell divisioncycle associated 3 [Source: HGNC Symbol; Acc: HGNC: 14624] CENPO 7centromere protein O [Source: HGNC Symbol; Acc: HGNC: 28152] FEN1 7 flapstructure-specific endonuclease 1 [Source: HGNC Symbol; Acc: HGNC: 3650]FOXM1 7 forkhead box M1 [Source: HGNC Symbol; Acc: HGNC: 3818] KIAA15247 KIF2C 7 kinesin family member 2C [Source: HGNC Symbol; Acc: HGNC:6393] KPNA2 7 karyopherin subunit alpha 2 [Source: HGNC Symbol; Acc:HGNC: 6395] MYBL2 7 MYB proto-oncogene like 2 [Source: HGNC Symbol; Acc:HGNC: 7548] NEK2 7 NIMA related kinase 2 [Source: HGNC Symbol; Acc:HGNC: 7745] RANBP1 7 RAN binding protein 1 [Source: HGNC Symbol; Acc:HGNC: 9847] SNRPB 7 small nuclear ribonucleoprotein polypeptides B andB1 [Source: HGNC Symbol; Acc: HGNC: 11153] SPC24 7 SPC24, NDC80kinetochore complex component [Source: HGNC Symbol; Acc: HGNC: 26913]TACC3 7 transforming acidic coiled-coil containing protein 3 [Source:HGNC Symbol; Acc: HGNC: 11524] TBC1D31 7 TBC1 domain family member 31[Source: HGNC Symbol; Acc: HGNC: 30888] TK1 7 thymidine kinase 1[Source: HGNC Symbol; Acc: HGNC: 11830] ZNF695 7 zinc finger protein 695[Source: HGNC Symbol; Acc: HGNC: 30954] AURKA 6 aurora kinase A [Source:HGNC Symbol; Acc: HGNC: 11393] BIRC5 6 baculoviral IAP repeat containing5 [Source: HGNC Symbol; Acc: HGNC: 593] BLM 6 BLM RecQ like helicase[Source: HGNC Symbol; Acc: HGNC: 1058] C17orf53 6 chromosome 17 openreading frame 53 [Source: HGNC Symbol; Acc: HGNC: 28460] CBX3 6chromobox 3 [Source: HGNC Symbol; Acc: HGNC: 1553] CCNB1 6 cyclin B1[Source: HGNC Symbol; Acc: HGNC: 1579] CCNE1 6 cyclin E1 [Source: HGNCSymbol; Acc: HGNC: 1589] CCNF 6 cyclin F [Source: HGNC Symbol; Acc:HGNC: 1591] CDC20 6 cell division cycle 20 [Source: HGNC Symbol; Acc:HGNC: 1723] CDC45 6 cell division cycle 45 [Source: HGNC Symbol; Acc:HGNC: 1739] CDCA5 6 cell division cycle associated 5 [Source: HGNCSymbol; Acc: HGNC: 14626] CDKN3 6 cyclin dependent kinase inhibitor 3[Source: HGNC Symbol; Acc: HGNC: 1791] CELSR3 6 cadherin EGF LAGseven-pass G-type receptor 3 [Source: HGNC Symbol; Acc: HGNC: 3230]CENPA 6 centromere protein A [Source: HGNC Symbol; Acc: HGNC: 1851]CEP72 6 centrosomal protein 72 [Source: HGNC Symbol; Acc: HGNC: 25547]CKS2 6 CDC28 protein kinase regulatory subunit 2 [Source: HGNC Symbol;Acc: HGNC: 2000] COL10A1 6 collagen type X alpha 1 chain [Source: HGNCSymbol; Acc: HGNC: 2185] CSE1L 6 chromosome segregation 1 like [Source:HGNC Symbol; Acc: HGNC: 2431] DBF4 6 DBF4 zinc finger [Source: HGNCSymbol; Acc: HGNC: 17364] GINS1 6 GINS complex subunit 1 [Source: HGNCSymbol; Acc: HGNC: 28980] GPR19 6 G protein-coupled receptor 19 [Source:HGNC Symbol; Acc: HGNC: 4473] KIF18A 6 kinesin family member 18A[Source: HGNC Symbol; Acc: HGNC: 29441] KIF4A 6 kinesin family member 4A[Source: HGNC Symbol; Acc: HGNC: 13339] KIFC1 6 kinesin family member C1[Source: HGNC Symbol; Acc: HGNC: 6389] MCM10 6 minichromosomemaintenance 10 replication initiation factor [Source: HGNC Symbol; Acc:HGNC: 18043] MCM2 6 minichromosome maintenance complex component 2[Source: HGNC Symbol; Acc: HGNC: 6944] MCM7 6 minichromosome maintenancecomplex component 7 [Source: HGNC Symbol; Acc: HGNC: 6950] MRGBP 6 MRGdomain binding protein [Source: HGNC Symbol; Acc: HGNC: 15866] MTHFD2 6methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase [Source: HGNC Symbol; Acc: HGNC:7434] NCAPH 6 non-SMC condensin I complex subunit H [Source: HGNCSymbol; Acc: HGNC: 1112] NDC80 6 NDC80, kinetochore complex component[Source: HGNC Symbol; Acc: HGNC: 16909] NUDT1 6 nudix hydrolase 1[Source: HGNC Symbol; Acc: HGNC: 8048] RNASEH2A 6 ribonuclease H2subunit A [Source: HGNC Symbol; Acc: HGNC: 18518] RUVBL1 6 RuvB like AAAATPase 1 [Source: HGNC Symbol; Acc: HGNC: 10474] SGOL1 6 SHCBP1 6 SHCbinding and spindle associated 1 [Source: HGNC Symbol; Acc: HGNC: 29547]SNRPG 6 small nuclear ribonucleoprotein polypeptide G [Source: HGNCSymbol; Acc: HGNC: 11163] TIMELESS 6 timeless circadian regulator[Source: HGNC Symbol; Acc: HGNC: 11813] TRIP13 6 thyroid hormonereceptor interactor 13 [Source: HGNC Symbol; Acc: HGNC: 12307] TROAP 6trophinin associated protein [Source: HGNC Symbol; Acc: HGNC: 12327]UBE2C 6 ubiquitin conjugating enzyme E2 C [Source: HGNC Symbol; Acc:HGNC: 15937] WDHD1 6 WD repeat and HMG-box DNA binding protein 1[Source: HGNC Symbol; Acc: HGNC: 23170] AFP1 6 Alpha fetoprotein[Source: HGNC symbol; AccHGNC: 317] Genes are shown above with theirname according to the HUGO Gene Nomenclature committee at genenames.org;“Acc HGNC” refers to the HGNG accession number on genenames.org

A more detailed analysis was performed for breast cancer, whereindividual genes in the list of 120 above were screened to see whichgenes met a profile wherein: (i) expression was elevated in cancerrelative to matched normal tissues; and (ii) background expressionacross all normal tissues sampled is low (e.g. to find “high confidence”markers that work especially well for breast cancer). This subanalysisidentified 4 candidates, of which 2, ubiquitin conjugating enzyme E2 C(UBE2C) and collagen type X alpha 1 chain (COL10A1) show a significantamount of expression in breast invasive carcinomas in TCGA relative tonormal tissues and low background expression.

When this analysis was completed individually for each tumor typebesides breast, it revealed two additional genes, cadherin 6 (CDH6) andATP binding cassette subfamily C member 4 (ABCC4) which showspecifically elevated expression in kidney (kidney renal cell carcinomaand kidney renal clear cell carcinoma) and prostate (prostateadenocarcinoma), respectively.

Example 11.—Analysis of Sensitivity of Detection for Ex-Vivo PromoterApproaches

To determine a limit of sensitivity for ex-vivo promoter approaches,where cells are isolated from a patient via e.g. a blood draw, the cellsare transfected with a synthetic biomarker, and expression of thebiomarker is assessed to determine the presence of diseased cells, anexperiment was performed dosing FLuc-bearing-lentivirus-transduced naiveH1299 cells doped into a background of normal human PBMCs. Variousnumbers of transduced cells were doped into 5 million isolated humanPBMCs (half the approximate number of cells obtained from an 8 ml blooddraw), and the samples were processed and analyzed for luciferaseexpression. The results are presented in FIG. 24 (FIG. 24), whichestimates that the limit of detection using FLuc as a syntheticbiomarker using this approach is 3-10 “diseased” reporter-active cellsper 5 million normal cells.

Example 12.—Cancer-Activated DNA Constructs Differentiate Tumor-Bearingand Healthy Mice

The survivin-SEAP nanoplasmid (nDNA-Survivin-SEAP) was formulated within vivo JetPEI, a linear polyethylenimine derivative that was also usedin PoC experiments (FIGS. 16A-16D). DNA nanoplasmids, like DNAminicircles, eliminate bacterial backbones and the need for antibioticresistant genes as a transformation selected marker using a growthrestrictive non-coding, antisense RNA sequence. Sharing similarproperties of enhanced expression, DNA nanoplasmids are produced withoutusing a complex recombination event, thus, DNA nanoplasmids can achieverobust expression and can be manufactured at scale equivalent to regularDNA plasmids. Significant efforts were used to optimize and characterizethe formulations prior to in vivo application. Thus, thenanoplasmid/JetPEI polyplexes, and any ternary transfection complexesused at Earli, are characterized by size, charge and polydispersity. Theoptimization process also took into consideration the properties ofencapsidation efficiency, serum stability and the ability to protect thepayload against nucleases. Collectively, these optimizations provide asystem with enhanced sensitivity and transfection capabilities.

A formulation of 15 μg of nDNA-Survivin-SEAP with JetPEI was generated,characterized and was injected intravenously into animals with modesttumor burden. The results indicate that at the highest differentialpoint on Day 4 there was a 36-fold increase in SEAP levels overnon-tumor-bearing animals that were dosed with identical levels of theformulated nanoplasmids (FIG. 26, see JetPEI delivery). The sensitivitywas dramatically increased and the overall level of specificity, asassessed in the difference in signal to noise ratio was increased overten-fold. The decline of cancer-activated expression of SEAP in thelatter portion of the current study is likely due to cell division and aloss of the DNA templates from the nucleus. Because multiple timepointsshowed an elevation of SEAP levels, an AUC analysis was performed foreach mouse. The AUC across all days provided 19-fold differential thannon-tumorigenic animals dosed with the nanoplasmids. Given the tightnessof intragroup data for each cohort and the clear differentiation in SEAPlevels, it was not surprising that the ROC analysis yielded 100%sensitivity and 100% specificity. DNA delivery using relatedpoly(β-amino ester)s C32-122 and C32-145 (see e.g., Mol Ther. 2007 July;15(7):1306-1312. doi: 10.1038/sj.mt.6300132 for structures) was alsoshown; such delivery agents had somewhat less efficient delivery thanJetPEI (FIG. 26A) but somewhat lower toxicity (FIG. 26F).

Example 13.—Assess Cancer-Activated DNA Constructs in Different TumorModels

To avoid complexities associated with assessment of tumor size usingbioluminescent-based techniques, tumor models in which disease burdencan be determined via caliper measurements including subcutaneous tumorsor models in which disease burden presents near the external surface ofthe animals are used. At least two orthotopic models are studied wherethe tissues are accessible to physical measurements with calipers.Characterization of each of the cell line used in the patient derivedxenograft (CDX) is performed prior to the establishment of each model.Additional validation is performed by in vitro transfection to confirmSEAP expression form the cancer-activated DNA constructs. When possible,tumorigenic models with intact immune systems are used.

The activity of biomarker expression from nDNA-Survivin-SEAP using asyngeneic mammary tumor model established by injecting 1×10⁶ 4 T1 cellsinto the mammary fat pad of Balb/c nude mice is initially characterized.The formulated vector is administered by intravenous injection intocohorts of tumor-bearing animals when the tumors attain sizes of either20 mm³, 50 mm³, 100 mm³ or 200 mm³. Plasma is collected from animals bysubmandibular bleeds at day −2 (pre-injection) as well as day 2, 6, 9,13 post-injection and will be processed for SEAP quantification and AUCand ROC analyses from all in vivo studies. Based on the precision of thedata in FIG. 25, each cohort is comprised of five animals. The number ofanimals per cohort is increased to 10 or more to account for thepotential loss in precision and differentiation of SEAP signal.

At terminal necropsy, tissues with high levels of the vector but lowlevels of transcript are subjected to bisulfate sequencing in order todetermine methylation status of the nanoplasmid DNA in those tissues. Inaddition, any organ that shows high levels of vector DNA or SEAPtranscript will be collected in repeat experiments in order to determineif gross histologic changes are present, via an external third-partyhistology (Histowiz, Brooklyn, N.Y.). Collectively, these data informIND-enabling GLP toxicology and biodistribution studies.

Example 14.—Develop New DNA Delivery Agents for In Vitro Transfection

The challenges of systemic gene delivery provide the guiding principlesfor the development of new formulations. Nucleic acids need to beefficiently encapsulated into nanoparticles to protect the cargo fromnuclease degradation. Once administered, the DNA nanoparticles may avoiddetection and destruction by innate or adaptive immunosurveillance inthe bloodstream. By a process known as extravasation, the nanoparticlesare taken up by tissues, including the cancer cells, where the DNAtemplate get released from delivery materials and be transported intothe nucleus to act as a transcriptional template. Finally, thecomponents of the nanoparticle shell are metabolized and eliminated fromthe cells without causing systemic toxicity. While these challenges havelong confounded efforts to successfully develop non-viral DNA deliverytechnologies, there have been significant advances in the cytosolicdelivery of RNA. This has been enabled by a new generation of ionizablecationic materials—both polymeric and lipid-based—that have superiorcell uptake and endosomal escape profiles and have culminated in theregulatory approval and commercialization of the first RNAi drug(Alnylam's Patisiran). The development strategy is to leverageimprovements in nucleic acid complexation, particle surface engineering,and endosomal escape, while conferring additional functionalities toimprove nuclear transport, to achieve non-viral DNA delivery atdiagnostically relevant concentrations for cancer detection.

For the development of new carriers for DNA nanoparticles, a two-prongedapproach is taken. The first is based on making modifications to lipidnanoparticle (LNPs) formulations that have been developed usingionizable cationic lipids that have been used for the delivery ofnucleic acids in gene therapy applications. LNPs are very efficient atcondensing anionic DNA and show high encapsulation efficiency and havemost often been applied to a wide variety of cargos for the transfectionof hepatic tissues. However, ionizable lipid structure and degradationprofiles can be engineered for distribution to other tissues. Becausethe inherent properties of size and charge are suitable forextravasation into the liver, the surface of these particles aremodified with polymers to prolong circulation.

The second prong uses of novel polymer compositions with uniquebiodegradable properties. Designed with hydrolysable bonds, polymerscalled poly (β-amino esters (PBAEs) have demonstrated an efficientability to transfect a wide variety of cells and are easily eliminatedfrom the cell and body once their cargo has been delivered. DNAformulated in PBAE complexes coated with polyglutamic acid were able toefficiently target and transduce T cells in vivo. PBAE/DNA polyplexesusing the new polymer subunits were prepared. These new PBAEsdemonstrated significantly less toxicity than JetPEI (FIGS. 26B-F).Other classes of degradable cationic polymers are studied such as chargealtering reversible transporters.

Clearance from the bloodstream by innate immunity through toll-likereceptor activation, liver clearance, macrophage uptake or eliciting anadaptive immune response through neutralizing antibodies is a challengeto in vivo delivery. As a result, strategies which shield the surfacecharge of formulated particles to increase the persistence and theability of complexes to be broadly distributed are investigated. Coatingthe nanoparticles with various ratios of covalently attachedpolyethylene glycol (PEG) is a strategy that is utilized. Methods toabsorb polyglutamic acid (PGA) onto the surface of nanoparticles throughelectrostatic interactions are also developed. Yet, these coatings cansubstantially increase the particle size as well as reduce cell uptake,which can both compromise transfection efficiency. As a result,modifications are being considered to the subunits which may degrade thestealth coating over time or may make de-shielding on the nanoparticlesin response to properties of the tumor microenvironment including lowpH, hypoxia or local proteases.

The DNA-delivering capability is increased of LNPs and polymericnanoparticles by including specific nuclear-targeting moieties that canretain DNA compaction and enhance nuclear localization by passivediffusion or directed transport. One such potential nuclear deliveryagent is protamine, which is comprised of arginine rich sequences thatbind DNA in a non-specific manner and is believed to act as a histonesubstitute for stabilization of DNA during sperm head condensation. Theaddition of protamine may generate delivery complexes that aresignificantly more compact. As can be seen in FIG. 27, thepre-incubation of DNA with protamine, resulted in the production ofmodestly smaller lipid nanoparticle complexes. It remains to bedetermined if the relatively smaller particles have enhancedtransduction properties. Incorporating a microtubule-associatedsequences (MTAS) and nuclear localization signals (NLS) into theformulations disclosed here may also be used to enhance nucleartransport.

A rigorous set of in vivo physical characterization methods areemployed, including understanding the encapsulation efficiency, size,charge and heterogeneity of the formulations, and stability in serumcontaining physiological buffer, in order to assess the quality of ourformulations prior to testing biodistribution and efficacy in animalmodels. The parameters for the polyplexes may include: (1) sizes rangingfrom 75 nm-150 nm (2) a net neutral charge or slightly negative surfacecharge (3) possess a polydispersity index of less than 0.15 (4) have anefficiency of encapsulation of greater than 85% and (5) serum stabilitygreater than 30 minutes.

Initial in vivo testing for biodistribution and transfection strength isperformed with formulated complexes of DNA nanoplasmids which utilizethe strong constitutive CMV promoter to drive the expression of fireflyluciferase (FIG. 28). Batches of approximately 20 different formulationsare tested per experiment. Cohorts of Balb/c mice (n=3 per cohort) areinjected with formulated complexes containing 25 μg of the DNAnanoplasmid. Blood samples taken at intermediate timepoints are used forcomprehensive clinical chemistry, whereas terminal bleeds are used forhematology as well as cytokine analysis to define the toxicity profileof the nanoparticle formulations that produce high levels ofluminescence in vivo or ex vivo. Further, animals with higher levels ofluminescence have additional organs collected for a more extensivebiodistribution analyses by QPCR for estimation of DNA vector copynumbers. Additional tissues to be collected include brain, gonads,injection site and bone marrow.

Formulations with broad distribution and luminescence equivalent to orsuperseding the levels provided by the control JetPEI group are movedinto efficacy studies. As with biodistribution and safety studies, thenew polyplexes may demonstrate efficacy that is similar or is betterthan the same nanoplasmid formulated with JetPEI. The use of the newformulations may also result in low background levels of SEAP innon-tumor bearing animals.

Example 15.—Scaling the Cancer-Activated Nanoplasmid Platform intoLarger Animal Models

While many oncology-based drugs have been tested and validated with thecontext to explore tumor xenografts established in murine models, thesesystems have inherent limitations including a combination of one or moreof the following: a lack of genetic diversity and a suitable tumormicroenvironment, a heterogeneity between tumor types regarding enhancedpermeability and retention (EPR) as well as the propensity of many, butnot all, of these models to be established in animals with severelycompromised immune systems or completely lacking an immune system.Moving the platform into naturally occurring cancers in larger animalmodels obviates many of these limitations. Likewise, one challenge withthe use of murine xenograft models is trying to comprehend the scalingof safe and efficacious dosing, particularly when considering theenormous difference in scale between a young murine species that weighs20-35 grams and the average weight of a human adult in North Americaexceeds 65 kilograms. In translating our platform into canine models, awide variety of naturally occurring types of tumors are evaluated in adiverse range of animal sizes, depending upon the breed of dog.

As to the nature of the test article, the initial studies are performedwith a construct comprised of the human survivin promoter driving theexpression of the human SEAP protein. The degree of identity between thesequences in the human and canine survivin promoter is conserved.Transient transfection of the nDNA-Survivin-SEAP construct into cellsderived from canine cancers indicate that the promoter is functional ina wide variety of canine cells (FIGS. 29A and 29B).

For the in vivo efficacy studies, only dogs with malignant tumors willbe enrolled. Information are collected on the dogs age, size, weight,breed as well as current treatments (if any). If available, the clinicsare also providing additional information on current treatments, tumorstaging, concomitant radiological data. For the initial studies, animalsthat have high existing burden of disease and are treatment naïve areenrolled, though a desire for expeditious treatment may require us torelax requirements on the latter. Three dosing groups with n=3 dogs percohort are injected with a formulated cancer-activated nanoplasmid viaan intravenous route of administration. Three additional dogs that maybe terminally ill from non-cancerous diseases may be used as controls ifthey can be enrolled, though it may prove difficult to ascertain thatthe animals are truly cancer-free. A blood draw covering clinicalchemistry and hematology will be taken just prior to dosing as well as1, 2, 4, and 6 hours post dosing for PK data. Additional blood sampleswill be collected 3, 6, 9, 12 and 15 days after dosing to monitor forclinical chemistry as well as detect the presence of the human SEAP inplasma. Additional blood parameters may be collected based on theresults of the acute toxicity studies. When possible, clinicians collectsamples from a subsequent biopsy or surgical resection of the tumorafter the administration of the disclosed test article and pass alongthe materials. Following nucleic acid isolation, the samples for thepresence of nanoplasmid DNA QPCR and for the presence of the SEAPtranscript by RT-QPCR are interrogated. RT-QPCR are used to assess therelative levels of survivin transcript within the tumors.

Example 16.—an Ex Vivo Approach for the Use of Cancer-ActivatedSynthetic Biomarker Constructs

As a general tool for large-scale detection of early cancers, thedevelopment costs could be significantly reduced, and timelinesappreciably shortened if we had the capability of employing thecancer-activated synthetic biomarker expression system in an ex vivosetting. A number of recent reports have been published indicating thatlive circulating tumor cells (CTCs) could be isolated and expanded exvivo, maintaining biological function unique to those cells thus makingit possible to apply the disclosed cancer-activated synthetic biomarkerplatform to these cells. The number of these cells that are shed intothe blood might be small.

Ex vivo application of the cancer-activated synthetic biomarker platformmay not be limited to trying to take advantage of dysregulatedexpression in CTCs. A number of groups have identified blood-basedbiomarkers of cancer by comparing the gene expression profiles fromPBMCs isolated from breast cancer patients versus expression profilesfrom healthy volunteer blood or from whole blood of lung cancerpatients. In addition, a cell-based in vivo sensor for activated M2macrophages that had been engineered with a tumor-activatable syntheticbiomarker expression system has been reported. Collectively, at least ata transient level, non-tumorigenic cells in the same milieu as cancercells may have transiently altered transcriptional profiles that may beexploited in an ex vivo approach using the disclosed cancer-activatedbiosynthetic marker platform. Collectively, any cell population,including CTCs, that may have an altered gene expression profile as aresult of cancer can be referred to as a “Transcriptionally AlteredCell” or TAC for short.

The cellular portion of whole blood, i.e. the PBMCs and other cellspresent, can be interrogated with the disclosed cancer-activatedbiomarker constructs by using recombinant adenoviruses or lentiviruseswith broad tropism for efficient delivery. Many different cell types areamenable high efficiency gene transfer with recombinant virusesresulting in the ability to introduce dozens or even hundreds of copiesof each transcriptional unit on a per cell basis. Each of thosetranscriptional units may drive the expression of analytes, such asluciferase, with high sensitivity levels that can be easily quantified.

In addition to highly sensitive reporters, the ability to efficientlytransduce a wide range of cell types should confer enhanced sensitivity,as measured by increased signal output, since efficient transductioneliminates the need to enrich specific subtypes of cells or rarepopulations such as EpCAM-positive CTCs.

To ascertain the theoretical sensitivity of an ex vivo assay, H1299cells that were engineered to constitutively express the fireflyluciferase (FLuc) protein, were spiked into 5e6 PBMCs from healthyvolunteers, the approximate number of cells from a standard 8 mL blooddraw. Following processing, a linear relationship of luciferaseexpression to spiked H1299s was noted with a detection limit between3-10 cells (FIG. 24). In order to understand if our viral vectors couldtransduce cancer cells in a complex mixture of human PBMCs, arecombinant adenovirus containing the human survivin promoter to driveexpression of firefly luciferase (Ad-Survivin-FLuc) was used totransduce a mixture increasing number of naïve (unmanipulated) H1299cells in the context of 5e6 PBMCs from healthy volunteers. Derived froma human non-small cell lung carcinoma cell line, H1299 cells havedemonstrated high levels of survivin expression and thus should providerobust luciferase expression if transduced with our vector. Following a2-day incubation period, luciferase activity analyses demonstrated thatthe sensitivity of the ex vivo assay can detect as few as 2 of the naïvecells spiked into 5e6 human PBMCs (FIGS. 30A and 30B) indicating theability to transduce and detect cancerous cells with this platform incomplex mixtures. In order to ascertain specificity, samples containingonly PBMCs were transduced with Ad-CMV-FLuc (FIG. 30C, blue column)resulting in robust luciferase expression and demonstrating that cellswithin the PBMC fraction are capable of being transduced by adenovirus.The lack of measurable luciferase activity from PBMCs transduced withAd-Survivin-FLuc (FIG. 30C, red column) versus non-transduced PBMCs(FIG. 30C, gray column) indicates that the vector only produces a robustsignal in the presence of the cells with dysregulated survivinexpression, such as the H1299 cells spiked into the PBMCs in FIG. 31B.

Similar experiments were performed in which the same Ad-Survivin-Flucvector was used to transduce samples containing 5×10⁵ canine PBMCs thathad been spiked with naïve canine cancer cells derived from dog tumors.High levels of canine survivin expression have also been previouslyreported in canine tumors including osteosarcoma. Although the survivinpromoter used in the vector was of human origin, the similarity betweenthe promoter sequences from the two species resulted in robustproduction of the luciferase reporter. Sensitivity for detection ofcanine tumor cells was down to the single cell level (FIG. 29A) anddetected cells derived from multiple canine tumor types (FIG. 29B).Finally, an initial set of human PBMCs derived from normal healthyvolunteers as well as from cancer patients were procured from commercialsources. Following transduction with the Ad-Survivin-Fluc construct, thesamples were incubated over 72 hours, lysed and assay for luciferaseactivity (FIG. 31). Normal PBMCs healthy human volunteers set thebaseline, while the individual cancer-derived patient samples hadelevated luciferase levels. Surprisingly several of the samples withstatistically significant data were collected from patients with Stage 1and Stage 2. The results with this experiment were obtained as a proofof concept study and there are parameters that must be optimized, suchas using an alternative promoter or combinations of promoters to impactthe specificity and rate of detection.

Example 17.—Canine Studies Using the First-Generation Ex Vivo AssayDemonstrates 89% Detection Rate of Lymphoma

Peripheral blood mononuclear cells (PBMCs) were separated from caninewhole blood using a BD Vacutainer® CPT™ mononuclear cell preparationtube (BD Biosciences, 362753). Separated PBMCs were resuspended in RPMImedium (ATCC, 302001) supplemented with heat-inactivated fetal bovineserum (VWR, 89510-184), penicillin-streptomycin antibiotic (Gibco,catalog 15140-122), and L-glutamine (ThermoFisher, 25030081). Cellviability was determined by trypan blue dye exclusion (Gibco, T10282).The ex vivo bioassay was performed in the following manner: ˜3×10⁶ PBMCswere seeded into a single well of a 96-well plate (VWR, 10861) in afinal volume of 200 microliters. Cells were then transduced at a MOI of0.3 with an adenoviral vector engineered to contain the fireflyluciferase reporter gene driven by the BIRC5/Survivin promoter.Transduced cells were allowed to incubate at 37° C. (5% CO2) for 72 hrs.Afterwards, medium was removed, and cells were lysed in the well with 20microliters of passive lysis buffer (Promega, E1910). The lysate wasthen transferred into a single well of a 96-well white solid assay plate(Corning, CLS3912), followed by the addition of 100 microliters ofluciferase substrate buffer (Promega, E4550). Luminescence was measuredat 0.3 second integration time using a Promega GloMax Navigatormicroplate reader. All data were collected and analyzed with GraphpadPrism. FIG. 32A shows a comparison of the fold-change in luminescenceexpression of healthy canine individuals (n=31) and canine lymphomacancer patients (n=17). FIG. 32B shows a diagnosis predictive capacityof survivin-activated luciferase activity to distinguish canine lymphomacancer subjects and healthy canine subjects.

Example 18.—Development of Combinatorial Cancer Detection Approach UsingCombinatorial Detection of Cancer-Activated Promoters

The cascades of regulatory sequences that drive the expression of thosedysregulated genes can be coupled into the cancer-activated biosyntheticmarker platform to drive the specificity and sensitivity required forimproved cancer detection rates with a high degree of accuracy beyondsimply looking for the gene product alone.

New promoters were identified by applying a bioinformatics approach tocompliment previously published literature on dysregulated geneexpression in malignancies. For the former, an analysis of TCGA isinitiated, which has curated over 20,000 primary cancer samples, from 33types of cancer against pair matched normal tissues. A wide variety ofother databases, such as the ICGC and the Clinical Proteome TumorAnalyses Consortium (CPTAC), may also be used.

The workflow permits using a series filters, including a slider bar toselect custom levels and threshold of signal to noise ratios acrossspecific tumor types and matched normal controls. The ability to filteron tumor staging is critical since dysregulated gene expression patternsthat occur in early stage cancers is studied first. Lastly, abiodistribution filter may help predict the relative contribution ofleaky promoters within the context of the different types oftransfection agent formulations disclosed herein.

The validity and rationale of many of the steps were tested by applyingthe filters independently against the TCGA database and assessing thetarget output versus published literature. In preliminary data,threshold filters of signal-to-noise ratios in cancer vs matched normaltissues were applied and the stringency progressively increased. Fornormal tissue expression data, Genotype-Tissue Expression (GTEx)database data was selected as an indicator of healthy tissue than thematched normal tissue data in the TCGA. Results from this step revealedoverexpression of 88 genes across a substantial number of tumor typesversus normal tissues. For validation of cancer-activated promoteractivity, the regulatory sequences from each of newly identifiedpromoter targets were: (a) subcloned into a nanoplasmid expressionconstruct in such a way that they are operably linked to a reporter openreading frame (e.g. a luciferase gene) to produce a promoter-reporterconstruct; and (b) transfected into various cancer cell lines.Specifically, several of the individual promoter sequences weresubcloned upstream of the cloning sequences of the firefly (Photinus)luciferase reporter gene and firefly luciferase biomarker expression wasanalyzed after each vector was transfected into a cancer-derived tissueculture cell line. A separate expression plasmid utilizing the CMVpromoter (a strong constitutive promoter) to drive expression of theorthogonal Renilla luciferase was added into the transfection mixture ata concentration 50-fold less than the test plasmid as an internalcontrol (assessment of the level of Renilla luciferase produced from theco-transfected plasmid permitted normalization of transfectionefficiency between different cell lines).

The cell lines utilized for transfection comprised: (a) immortalizedcell lines selected with the guidance of expression data from the CancerCell Line Encyclopedia (CCLE); (b) primary patient human cell linesestablished from tumors of different origin (e.g. lung, breast andpancreatic tissues); and (c) canine cell lines isolated from varioustypes of dog/canine cancers. In vitro transfections of each cell linewere performed using Lipofectamine 3000 as a transfection agent(although the disclosed cassettes are also readily transferrable intorecombinant expression constructs for packaging into recombinant viralvectors such as adenovirus). The relative levels of each gene wereconfirmed by assessment of firefly (Photinus) luciferase activity, afternormalization for transfection using the Renilla luciferase signal. Theactivity of each of the reporter constructs in cell lines derived fromcancers of the liver, ovaries and pancreas is shown in FIGS. 33A, 33Band 33C respectively, wherein the relative intensity of expression ofeach reporter-promoter construct in each cell line is depicted via colordensity (particular values are shown with reference to the densitylegend to the right of each graph). Likewise, the activity of thereporter-promoter constructs in cell lines derived from breast and lungis shown in FIGS. 33D and 33E respectively. The same data was generatedby transfection of the promoter-reporter construct into untransformedcell lines derived from normal lung tissues; the corresponding lowlevels activity of the same promoters in these untransformed cells (FIG.33F) demonstrates that many of these cancer-activated promoters may beuseful in differentiating tumors from non-tumor bearing tissues (seee.g., MMP12, CEP55, COL10A1, KIF20A, FAM111B, CST1, AFP, BIRC5, UBE2C,MCM10) Finally, the activity of the constructs were tested in cell linesderived from canine cancers (FIG. 34). Despite the promoter sequenceshaving been derived from human sequences, many of the constructs yieldedrobust activity in the canine cell line (see e.g., BIRC5, BIRC5-501,CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, andcBIRC5).

Example 19.—Development of a Multiplex Nucleic Acid/Protein BarcodeApproach Combining Cancer-Activated Promoters to Improve CancerDetection Rates

The use of easily quantifiable synthetic barcodes as surrogate reportermarkers of activity can simplify analyses where multiple outputs are tobe analyzed (e.g., simultaneous analysis of the activity of multiplepromoters in a cell line). In order to assess if the simultaneous use ofmultiple unique promoters (i.e. multiplexing) would be amenable to theuse of nucleic acid or protein barcodes, a series of individualpromoters were first cloned upstream a of secreted luciferase molecule.The secretory domain/signal peptide from the IL-6 protein was includedat the termini of the protein, resulting in the conversion of luciferase(normally restricted to intracellular localization) into an enzyme thatis secreted from the cells. Subsequently, multiple DNA constructs weregenerated in which a series of unique nucleic acid barcodes wereintroduced in between the sequences encoding for the signal peptide andthe luciferase (FIG. 35A, see colored inserts between signal peptide andluciferase). In this case, barcodes of 6 nucleotides were used (whichcan provide up to 4096 unique sequences for detection) though longerstretches of nucleotides (e.g. at least 6, at least 7, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, or at least 20or more) are envisioned and provide for a larger set of combinations.Because each of the different promoters is assigned to a correspondingunique barcode due to inclusion in the expressed protein construct, oneshould be able to quantify the levels of barcode as a surrogate markerfor reporter expression. These signal peptide-barcode-luciferaseconstructs were then subcloned into nanoplasmid constructs under thecontrol of several candidate reporters such as ABCC4, BIRC5 (akasurvivin), UBE2C, COL10A1, E2F, FOXA1 and MCM10 and were transfectedinto cell lines for expression analysis (see e.g., FIG. 36).

Each of the barcoded, cancer-activated luciferase reporter constructswas tested individually by transfecting parallel plates of H1299 cells.A DNA construct that contained a CMV-driven Renilla luciferaseexpression cassette was included into each transfection mixture tocontrol for variances in transfection efficiency. Following incubationfor an additional 24 hours post transfection, the cells were lysed andassayed for luminescence activity. FIG. 35B demonstrates that, whentested individually, each of the promoters produces discernable levelsof luciferase activity within the H1299 cells. Furthermore, thedifferences in relative strength of each of the promoters results in aunique pattern. In a parallel set of plates, H1299 cells weretransfected with a mixture of the barcoded constructs containingequimolar ratios of each of the cancer-activated reporter constructs.Following a 24-hour incubation of the transfected cells, RNA waspurified from the cells and analyzed by next generation sequencing toquantify the relative levels of barcoded sequences present within thecellular transcripts. FIG. 35C demonstrates that transfection of theH1299 cells with the multiplexed mixture resulted in a nearly identicalpattern of relative expression pattern as the individually transfectedcells as assessed by the quantities of the nucleic acid barcode. Theresults from these combined experiments provide evidence that theseunique cancer-activated expression patterns from the promoters, orfingerprints, may have utility in identifying molecular characteristicsassociated with the specific cells that are being tested. Furthermore,barcoding can provide a useful surrogate for assessing the relativeactivity levels in a multiplex format.

Example 20.—Use of an Antibody Capture Format to Detect Activation ofPromoter-Reporter Constructs in Cells

The types of barcodes used to identify unique promoter/reportercombinations may exist as nucleic acid sequences (FIG. 35A).Alternatively, barcodes may take other forms including, but not limitedto, the use of small proteins or peptide fragments. In this currentexample, the peptide barcode fragment that is fused onto the termini ofthe luciferase reporter protein allows the secreted luciferase to becaptured in an enzyme-linked immunosorbent assay (ELISA) based assay inwhich the each of the individual wells of the 96-well plate used toperform the assay are first coated with a specific antibody withaffinity to one of the unique protein tags. In this way, each well inthe plate can be used to pan and surveil the complex mixture of secretedmedia for the presence of individual peptide tags. To demonstrate theutility of this approach, an engineered secreted form of a luciferaseprotein was generated by fusion of the secretory domain/signal peptidefrom IL-6 to the N-terminus of luciferase. Additionally, a series ofpeptide epitope-based barcodes were introduced in between the sequencesencoding for the signal peptide and the luciferase (FIG. 36A). Epitopetags included for the initial proof of concept experiments included FLAG(DYKDDDDK), HA (a fragment of the influenza hemagglutinin protein), V5(a peptide domain present on the V and P proteins from the paramyxovirusknown as simian virus 5) and HSV (a fragment of a protein expressed bythe herpes simplex virus). Using the same CMV promoter, the fourconstructs with unique protein barcodes were individually transfectedinto cells (see e.g., FIG. 36A). After an additional 24 hours ofincubation, aliquots of media were collected from each transfectioncondition and panned in a 96-well plate in which each of the individualwells were coated with a unique capture antibody against each of theunique epitopes (FIG. 36B). Following a series of buffer washes toeliminate non-specific binding, each of the wells was developed forluciferase activity. The data, shown in FIG. 36C, demonstrates thatH1299 cells transfected with a construct harboring a specific proteinepitope could be captured in wells coated with the correspondingantibodies—as demonstrated by the large increase in luciferase activity.Conversely, that same lysate does not result in an increase inluminescence in non-antibody coated control wells (blocked fornon-specific binding) or in wells coated with antibodies against otherepitopes. Taken together, these data demonstrate that the barcodes canprovide a physical method of separation in addition to providing asurrogate measure of relative promoter activity.

The peptide fragment used as a barcode can be designed for alternativescreening and panning methodologies other than an ELISA format.Alternative assay formats may be useful to provide fast, high qualityand inexpensive solutions for the detection of specific analytes. As oneexample, a rapid test using lateral flow chromatography with patienturine has been commonly employed for the detection of human pregnancy.In order to test the ability of alternative assay formats for thedetection of cancer-activated markers, the nucleic acid barcodesinserted into our secreted variant of luciferase was replaced with thebeta subunit from the human chorionic gonadotropin (hCG) protein. Thechimeric protein was cloned downstream of either the cancer-activatedhuman survivin promoter or from the CMV promoter (FIG. 37A). In order toensure that the hCG barcode did not alter the activity of theconstructs, parallel sets of plates of H1299 cells were transfected withrecombinant DNA constructs. Identical DNA constructs lacking the hCGbarcode served as controls. A DNA construct that contained a CMV-drivenRenilla luciferase expression cassette was included into eachtransfection mixture to control for variances in transfectionefficiency. Following incubation for an additional 24 hours posttransfection, the cells were lysed, and aliquots of the media wereassayed for luminescence activity. As seen in FIG. 37B, the presence ofthe hCG bar code did not substantially alter the expression of theluciferase reporter protein. Finally, 700 ul of the supernatant of thesame was loaded into a commercial pregnancy test kit, bought at a localpharmacy. Seen in FIG. 37, the pregnancy test kit can clearlydifferentiate between cells which had been transfected with anon-barcoded variant of the reporter versus the hCG barcoded variant.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

EMBODIMENTS

The following are intended to be example embodiments and not limiting inany way.

1. A method comprising:

-   -   (a) administering to a subject a composition, wherein said        composition induces expression of a biomarker in a diseased cell        preferentially over expression of said biomarker in non-diseased        cells in said subject such that a relative ratio of said        biomarker expressed in said diseased cell over said non-diseased        cells is greater than 1.0;    -   (b) detecting said biomarker; and    -   (c) using said biomarker detected in (b) to determine that said        subject has said diseased cell at an accuracy of at least 70%.        2. The method of embodiment 1, wherein said relative ratio is a        concentration ratio.        3. The method of embodiment 1 or 2, wherein said biomarker is        detected in biological sample from said subject.        4. The method of embodiment 3, wherein said biological sample is        a bodily fluid from said subject.        5. The method of embodiment 4, wherein said biological sample is        a blood or blood-based sample from said subject.        6. The method of embodiment 3, wherein said biological sample is        a gaseous sample from said subject.        7. The method of embodiment 6, wherein said gaseous sample is        the breath from said subject.        8. The method of any one of embodiments 1-7, wherein said        subject is a mammal.        9. The method of embodiment 8, wherein said subject is human.        10. The method of embodiment 8, wherein said subject is an        animal.        11. The method of embodiment 3, wherein said biological sample        is measured in situ within the human or animal body.        12. The method of any one of embodiments 1-11, wherein said        composition comprises a nucleic acid vector.        13. The method of embodiment 12, wherein said vector is selected        from the group consisting of nanoplasmids, plasmids,        minicircles, recombinant viral vectors, or CELiDs.        14. The method of embodiment 13, wherein said composition        comprises a minicircle, wherein said minicircle is a        self-replicating minicircle.        15. The method of embodiment 14, wherein said self-replicating        minicircle comprises an S/MAR element.        16. The method of any one of embodiments 1-15, wherein said        composition comprises a promoter operably linked to a nucleotide        sequence encoding the biomarker.        17. The method of embodiment 16, wherein said promoter drives        expression in a plurality of different types of diseased cells        in said subject.        18. The method of any one of embodiments 1-17, wherein said        promoter drives expression of said biomarker in said diseased        plurality of different types of diseased cells preferentially        over expression of said biomarker in said non-diseased cells in        said subject.        19. The method of embodiment 17, wherein promoter is selected        from the group consisting of a Survivin promoter (BIRC5), a        CXCR4 promoter, an ATP binding cassette subfamily C member 4        (ABCC4) promoter, an anterior gradient 2, protein disulphide        isomerase family member (AGR2) promoter, activation induced        cytidine deaminase (AICDA) promoter, an UDP-GlcNAc:betaGal        beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a        cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5        (CEACAM5) promoter, a centromere protein F (CENPF) promoter, a        centrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3)        promoter, a claudin 4 (CLDN4) promoter, a collagen type XI alpha        1 chain (COL11A1) promoter, a collagen type I alpha 1 chain        (COL1A1) promoter, a cystatin SN (CST1) promoter, a denticleless        E3 ubiquitin protein ligase homolog (DTL) promoter, a family        with sequence similarity 111 member B (FAM111B) promoter, a        forkhead box A1 (FOXA1) promoter, a kinesin family member 20A        (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitotic        spindle positioning (MISP) promoter, a matrix metallopeptidase 1        (MMP1) promoter, a matrix metallopeptidase 12 (MMP12) promoter,        a matrix metallopeptidase 13 (MMP13) promoter, a mesothelin        (MSLN) promoter, a cell surface associated mucin 1 (MUC1)        promoter, a phospholipase A2 group IID (PLA2G2D) promoter, a        regulator of G protein signaling 13 (RGS13) promoter, a        secretoglobin family 2A member 1 (SCGB2A1) promoter,        topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD)        promoter, a ubiquitin conjugating enzyme E2 C (UBE2C), a USH1        protein network component harmonin (USH1C), a V-set domain        containing T cell activation inhibitor 1 (VTCN1) promoter, a        Hexokinase type II promoter, a TRPM4 promoter, a stromelysin 3        promoter, a surfactant protein A promoter, a secretory        leukoprotease inhibitor promoter, a tyrosinase promoter, a        stress-inducible grp78/BiP promoter, an interleukin-10 promoter,        an α-B-crystallin/heat shock protein 27 promoter, an epidermal        growth factor receptor promoter, a mucin-like glycoprotein        promoter, an mts1 promoter, an NSE promoter, a somatostatin        receptor promoter, a c-erbB-3 promoter, a c-erbB-2 promoter, a        c-erbB4 promoter, a thyroglobulin promoter, an α-fetoprotein        promoter, a villin promoter, an albumin promoter, a glycoprotein        A33 promoter, the B cell-specific Moloney leukemia virus        insertion site 1 promoter, a cyclooxygenase-2 promoter, a        fibroblast growth factor promoter; a human epidermal growth        factor receptor 2, a human telomerase reverse transcriptase        promoter; a kinase domain insert containing receptor promoter; a        rad51 recombinase promoter; TTF-1, an urokinase-type plasminogen        activator receptor promoter, a ubiquitin conjugating enzyme E2 T        (UBE2T) promoter, a checkpoint kinase 1 (CHEK1) promoter, an        epithelial cell transforming 2 promoter (ECT2), a BCL2-like 12        (BCL2L12) promoter, a centromere protein I (CENPI) promoter, an        E2F transcription factor 1 (E2F1) promoter, a flavin adenine        dinucleotide synthetase 1 (FLAD1) promoter, a protein        phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an        ubiquitin conjugating enzyme E2 S (UBE2S) promoter, an aurora        kinase A and ninein interacting protein (AUNIP) promoter, a cell        division cycle 6 (CDC6) promoter, a centromere protein L (CENPL)        promoter, a DNA replication helicase/nuclease 2 (DNA2) promoter,        a DSN1 homolog, MIS12 kinetochore complex component (DSN1)        promoter, a deoxythymidylate kinase (DTYMK) promoter, a G        protein regulated inducer of neurite outgrowth 1 (GPRIN1)        promoter, a mitochondrial fission regulator 2 (MTFR2) promoter,        a RAD51 associated protein 1 (RAD51AP1) promoter, a small        nuclear ribonucleoprotein polypeptide A′ (SNRPA1) promoter, an        ATPase family, AAA domain containing 2 (ATAD2) promoter, a BUB1        mitotic checkpoint serine/threonine kinase (BUB1) promoter, a        calcyclin binding protein (CACYBP) promoter, a cell division        cycle associated 3 (CDCA3) promoter, a centromere protein O        (CENPO) promoter, a flap structure-specific endonuclease 1        (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, a cell        proliferation regulating inhibitor of protein phosphatase 2A        (KIAA1524) promoter, a kinesin family member 2C (KIF2C)        promoter, a karyopherin subunit alpha 2 (KPNA2) promoter, a MYB        proto-oncogene like 2 (MYBL2) promoter, a NIMA related kinase 2        (NEK2) promoter, a RAN binding protein 1 (RANBP1) promoter, a        small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB)        promoter, a SPC24/NDC80 kinetochore complex component (SPC24)        promoter, a transforming acidic coiled-coil containing protein 3        (TACC3) promoter, a TBC1 domain family member 31 (TBC1D31)        promoter, a thymidine kinase 1 (TK1) promoter, a zinc finger        protein 695 (ZNF695) promoter, an aurora kinase A (AURKA)        promoter, a BLM RecQ like helicase (BLM) promoter, a chromosome        17 open reading frame 53 (C17orf53) promoter, a chromobox 3        (CBX30) promoter, a cyclin B1 (CCNB1) promoter, a cyclin E1        (CCNE1) promoter, a cyclin F (CCNF), a cell division cycle 20        (CDC20) promoter, a cell division cycle 45 (CDC45) promoter, a        cell division cycle associated 5 (CDCA5) promoter, a cyclin        dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF        LAG seven-pass G-type receptor 3 (CELSR3) promoter, a centromere        protein A (CENPA) promoter, a centrosomal protein 72 (CEP72)        promoter, a CDC28 protein kinase regulatory subunit 2 (CKS2)        promoter, a collagen type X alpha 1 chain (COL10A1) promoter, a        chromosome segregation 1 like (CSE1L) promoter, a DBF4 zinc        finger promoter, a GINS complex subunit 1 (GINS1) promoter, a G        protein-coupled receptor 19 (GPR19) promoter, a kinesin family        member 18A (KIF18A) promoter, a kinesin family member 4A (KIF4A)        promoter, a kinesin family member C1 (KIFC1) promoter, a        minichromosome maintenance 10 replication initiation factor        (MCM10) promoter, a minichromosome maintenance complex component        2 (MCM2) promoter, a minichromosome maintenance complex        component 7 (MCM7) promoter, a MRG domain binding protein        (MRGBP) promoter, a methylenetetrahydrofolate dehydrogenase        (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase        (MTHFD2) promoter, a non-SMC condensin I complex subunit H        (NCAPH) promoter, a NDC80, kinetochore complex component (NDC80)        promoter, a nudix hydrolase 1 (NUDT1) promoter, a ribonuclease        H2 subunit A (RNASEH2A) promoter, a RuvB like AAA ATPase 1        (RUVBL1) promoter, a serologically defined breast cancer antigen        NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated        1 (SHCBP1) promoter, a small nuclear ribonucleoprotein        polypeptide G (SNRPG) promoter, a timeless circadian regulator        promoter, a thyroid hormone receptor interactor 13 (TRIP13)        promoter, a trophinin associated protein (TROAP) promoter, a        ubiquitin conjugating enzyme E2 C (UBE2C) promoter, a WD repeat        and HMG-box DNA binding protein 1 (WDHD1) promoter, an alpha        fetoprotein (AFP) promoter, a fragment thereof, or any        combination thereof.        20. The method of any one of embodiments 1-19, wherein said        biomarker is selected from the group consisting of MRI reporter,        a PET reporter, a SPECT reporter, a photoacoustic reporter, a        bioluminescent reporter, a fluorescent reporter,        chemiluminescent reporter, luminescence reporter, colorimetric        reporter, a quantifiable nucleic acid biomarker, and any        combination thereof.        21. The method of embodiment 20, wherein the quantifiable        nucleic acid biomarker is an engineered miRNA.        22. The method of embodiment 20 or 21, wherein detection of said        biomarker determines a location of the diseased cell.        23. The method of any one of embodiments 1-22, wherein said        biomarker is detectable in a bodily sample of said subject, by        non-invasive imaging or combinations thereof.        24. The method of embodiment 23, wherein said biomarker is        detected using a blood-based assay.        25. The method of any one of embodiments 1-24, wherein said        composition further comprises a transfection agent.        26. The method of embodiment 25, wherein said transfection agent        is a linear or branched polyethylenimine, nanoparticle,        lipophilic particle, solid nanoparticle, peptide, micelle,        dendrimer, polymeric composition, hydrogel, synthetic or        naturally derived exosome, virus-like particles, or any        combination thereof.        27. The method of any one of embodiments 1-26, wherein said        composition further comprises a pharmaceutically acceptable        carrier.        28. The method of embodiment 27, wherein said pharmaceutically        acceptable carrier is selected from the group consisting of        water, peanut oil, soybean oil, mineral oil, sesame oil, saline,        gum acacia, gelatin, starch paste, talc, keratin, colloidal        silica, urea, aqueous dextrose, glycerol solution, glucose,        lactose, sucrose, glycerol monostearate, sodium chloride        solution, propylene, glycol, cocoa butter, or ethanol.        29. A method for treating a subject having or suspected of        having a disease, comprising administering to said subject a        composition that induces expression of a therapeutically        effective agent by a diseased cell associated with said disease        preferentially over expression of said therapeutically effective        agent by non-diseased cells in said subject such that a relative        concentration of said therapeutically effective agent expressed        by said diseased cell over said non-diseased cells is greater        than 1.0, which therapeutically effective agent treats said        subject at a therapeutic efficacy of at least 10% as determined        by a decrease in a cell population of said diseased cell.        30. The method of embodiment 29, wherein said composition        comprises a vector.        31. The method of embodiment 29 or 30, wherein said composition        comprises a promoter operably linked to a nucleotide sequence        encoding a therapeutically effective agent.        32. The method of embodiment 31, wherein said therapeutically        effective agent is selected from the group consisting of a        therapeutically effective polypeptide, small activating RNA        (saRNA), microRNA (miRNA), small interfering RNA (siRNA) or        combinations of polypeptide and said nucleic acids.        33. The method of embodiment 32, wherein said therapeutically        effective agent is selected from the group consisting of HSVtk,        cytosine deaminase, DT diaphorase, nitroreductase, guanine        phosphoribosyl transferase, purine nucleoside phosphorylase,        thymidine phorphorylase, carboxylesterase, folylpolyglutamyl        synthetase, carboxypeptidase A1, carboxypeptidase G2, cytochrome        P-450.        34. A composition comprising a first nucleic acid sequence        encoding a first polypeptide or nucleic acid biomarker and a        second nucleic acid sequence encoding a second polypeptide or        second nucleic acid biomarker, wherein said composition is        configured such that when said composition is in a cell: said        second polypeptide or nucleic acid biomarker is expressed in an        amount that reflects delivery of said first and said second        nucleic acids to said cell, and said first polypeptide or        nucleic acid biomarker is expressed differentially in a diseased        cell versus a non-diseased cell.        35. The composition of embodiment 34, wherein:    -   (i) said cell induces expression of said first nucleic acid        sequence in a diseased cell preferentially over expression of        said first nucleic acid sequence in non-diseased cells, wherein        said expressed first polypeptide or nucleic acid is a detectable        biomarker or a therapeutic agent; and    -   (ii) said cell induces expression of said second nucleic acid        sequence equally in diseased and in non-diseased cells and said        second nucleic acid sequence yields said second polypeptide or        nucleic acid biomarker that is not said detectable biomarker or        said therapeutic agent, such that a level of expression of said        second polypeptide or nucleic acid biomarker provides a control        for assessing the relative level of said nucleic acid sequences        in said cell.        36. The composition of embodiment 34 or 35, wherein in said        composition said sequences comprising said first nucleic acid        sequence encoding said first polypeptide and said second nucleic        acid sequence encoding said second polypeptide are on        independent genetic constructs.        37. The composition of any one of embodiments 34-36, wherein        said first polypeptide is said detectable biomarker and said        therapeutic agent.        38. The composition of any one of embodiments 34-37, wherein        said cell is a diseased cell.        39. The composition of any one of embodiments 34-38, wherein        said composition further comprises a vector comprising said        first and said second nucleic acid.        40. The composition of embodiment 39, wherein said vector        comprises:    -   (a) a first promoter operably linked to said first nucleic acid        sequence, wherein said promoter induces expression of said first        nucleic acid sequence in a diseased cell preferentially over        expression of said first nucleic acid sequence in non-diseased        cells; and    -   (b) a second promoter sequence that induces expression equally        in diseased and in non-diseased cells and is operably linked to        said second nucleic acid.        41. The composition of embodiment 39 or embodiment 40, wherein        said vector is a non-viral vector.        42. The composition of embodiment 41, wherein said non-viral        vector is a minicircle vector.        43. The composition of embodiment 39 or embodiment 40, wherein        said vector is a nanoplasmid vector.        44. The composition of any one of embodiments 34-42, wherein        said first and second polypeptides or nucleic acid biomarkers        are detectable in a bodily sample of said subject, by        non-invasive imaging or combinations thereof.        45. A method of detecting diseased cells in a subject,        comprising administering a composition to said subject, wherein        said composition comprises:    -   a first nucleic acid sequence encoding a first polypeptide or        nucleic acid biomarker and a second nucleic acid sequence        encoding a second polypeptide or second nucleic acid biomarker,        -   wherein said composition is configured such that when said            composition is in a cell:        -   (i) said cell induces expression of said first nucleic acid            sequence in a diseased cell preferentially over expression            of said first nucleic acid sequence in non-diseased cells,            wherein said first polypeptide is a detectable biomarker or            a therapeutic agent; and        -   (ii) said cell induces equivalent expression of said second            nucleic acid sequence equally in diseased and in            non-diseased cells and said second nucleic acid sequence            yields said second polypeptide that is not said detectable            biomarker or said therapeutic agent, such that a level of            expression of said second polypeptide provides a control for            assessing the relative level of said nucleic acid sequences            in said cell.            46. The method of embodiment 45, wherein in said composition            said sequences comprising said first nucleic acid sequence            encoding said first polypeptide or nucleic acid biomarker            and said second nucleic acid sequence encoding said second            polypeptide or nucleic acid biomarker are on independent            constructs.            47. The method of embodiment 45 or embodiment 46, wherein            said first polypeptide is said detectable biomarker and said            therapeutic agent.            48. The method of any one of embodiments 45-47, wherein said            cell is a diseased cell.            49. The method of any one of embodiments 45-48, wherein said            composition further comprises a vector comprising said first            and said second nucleic acid.            50. The method of embodiments 49, wherein said vector            comprises:    -   (a) a first promoter operably linked to said first nucleic acid        sequence, wherein said promoter induces expression of said first        nucleic acid sequence in a diseased cell preferentially over        expression of said first nucleic acid sequence in non-diseased        cells; and    -   (b) a second promoter sequence that induces equivalent        expression equally in diseased and in non-diseased cells and is        operably linked to said second nucleic acid.        51. The method of embodiment 49 or embodiment 50, wherein said        vector is a non-viral vector.        52. The method of embodiment 51, wherein said non-viral vector        is a minicircle vector.        53. The method embodiment 49 or embodiment 50, wherein said        vector is a nanoplasmid vector.        54. The method of any one of embodiments 45-52, wherein said        first and second polypeptides are detectable in a bodily sample        of said subject, by non-invasive imaging or combinations        thereof.        55. A composition comprising    -   a first nucleic acid sequence encoding a first polypeptide and    -   a second nucleic acid sequence encoding a second polypeptide,        -   wherein said composition is configured such that when said            composition is in a cell: (i) said cell expresses said first            nucleic acid sequence to yield said first polypeptide;        -   (ii) said cell expresses said second nucleic acid sequence            to yield said second polypeptide; and        -   (iii) said first polypeptide and said second polypeptide            expressed by said cell are configured to combine to form a            heterodimer protein.            56. The composition of embodiment 55, wherein said first            nucleic acid sequence and said second nucleic acid sequence            are operably linked to a first genetic element and a second            genetic element, wherein both said first genetic element and            said second genetic element are selectively activated to            express said first and said second polypeptide in a same            diseased cell type.            57. The composition of embodiment 56, wherein said first or            said second generic element is a promoter, an enhancer, or a            miRNA-binding site.            58. The composition of any one of embodiments 55-57, wherein            said heterodimer protein is an FRB/FKBP12 heterodimer, a            split luciferase protein, or a split GFP protein.            59. The composition of embodiment 58, wherein said            FRB/FKBP12 heterodimer linked to first and second halves of            a Cre recombinase.            60. The composition of any one of embodiments 55-59, wherein            said first and said second polypeptide are linked to a first            and a second autofluorescent protein, wherein said first and            said second autofluorescent protein form a FRET pair.            61. The composition of any one of embodiments 55-60, wherein            said heterodimer protein is configured to modulate the            activity of a diseased cell.            62. The composition of any one of embodiments 55-61, wherein            said first polypeptide and said second polypeptide are on            independent genetic constructs.            63. A method of detecting or treating a diseased cell,            comprising administering a composition to a subject            according to any one of embodiments 55-62, wherein said            first and said second polypeptide are selectively            transcribed or translated in said diseased cell.            64. The method of embodiment 63, comprising detecting said            heterodimer protein.            65. A composition comprising a non-naturally occurring            recombinant genetic construct comprising a sequence encoding            a polypeptide or nucleic acid sequence, and wherein said            sequence comprises a first promoter that selectively drives            expression of said polypeptide or nucleic acid biomarker            sequence in a plurality of different types of cells isolated            from a subject when transduced into said cells ex vivo.            66. The composition of embodiment 65, comprising said cells.            67. The composition of embodiment 65 or 66, wherein said            cells comprise cells found in blood or blood fractions,            saliva, urine, stool, cerebrospinal fluid, semen, vaginal            secretions, sputum, sweat, breast milk, synovial fluid,            mucus (including rheum), tears, bile, gastric fluid,            interstitial fluid, biopsies of tissues or epithelial cells            that are naturally shed or specifically collected from the            body (such as cheek cell scrapings), aqueous humor, amniotic            fluid, pleural fluid or breath exhalation.            68. The composition of any one of embodiments 65-67, wherein            said plurality of different types of cells are diseased or            disordered cells.            69. The composition of embodiment 68, wherein said diseased            or disordered cells are a plurality of different types of            tumor cells.            70. The composition of any one of embodiments 65-69, wherein            said first promoter is a pan-tumor specific promoter.            71. The composition of any one of embodiments 65-70, wherein            said first promoter is a cancer-specific promoter.            72. The composition of any one of embodiments 65-71, wherein            said first promoter is an endogenous promoter from said            cells activated when the cell is in the diseased state.            73. The composition of any one of embodiments 65-72, wherein            said recombinant genetic construct comprises retroviral,            lentiviral, or adenoviral packaging elements or long            terminal repeats.            74. The composition of any one of embodiments 65-73, wherein            said recombinant genetic construct is a non-viral vector.            75. The composition of embodiment 74, wherein said non-viral            vector is a minicircle vector.            76. The composition of any one of embodiments 65-75, wherein            the composition has a certain diagnostic efficiency, wherein            the diagnostic efficiency is measured in a diseased cell            preferentially over expression of said biomarker in            non-diseased cells in said subject such that a relative            ratio of said biomarker expressed in said diseased cell over            said non-diseased cells is greater than 1.0;            (b) detecting said biomarker; and (c) using said biomarker            detected in (b) to determine that said subject has said            diseased cell at an accuracy of at least 90%.            77. The composition of any one of embodiments 65-76, wherein            said polypeptide or nucleic acid sequence is a reporter            polypeptide and is selected from the group consisting of a            photoacoustic reporter, a bioluminescent reporter, an            autofluorescent reporter, a chemiluminescent reporter, a            luminescent reporter, or a colorimetric reporter, a            quantifiable nucleic acid biomarker, or any combination            thereof.            78. The composition of embodiment 77, wherein said reporter            polypeptide is a luminescent reporter, and said luminescent            reporter is luciferase.            79. The method of any one of embodiments 65-78, wherein said            polypeptide or nucleic acid sequence is a nucleic acid            sequence, and is a ribozyme, a self-splicing intron, a            microRNA, a RNA aptamer, or another type of quantifiable RNA            biomarker.            80. The composition of embodiment 79, wherein said nucleic            acid sequence biomarker is detectable by quantitative PCR,            sequencing, or hybridization-based techniques.            81. The composition of any one of embodiments 65-80, wherein            said cell is a diseased or disordered cell, wherein said            genetic construct further encodes a second polypeptide that            modulates the proliferation the diseased or disordered cell            diseased cell under the control of a second promoter that            selectively drives expression of said second polypeptide in            said diseased or disordered cell.            82. A method for detecting a diseased or disordered cell            ex-vivo, comprising delivering ex vivo a non-naturally            occurring recombinant genetic construct to a population of            cells isolated from a subject, wherein the non-naturally            occurring recombinant genetic construct comprises:    -   a sequence encoding a polypeptide or nucleic acid biomarker        sequence,    -   wherein said sequence comprises a first promoter that        selectively drives expression of said polypeptide or nucleic        acid biomarker sequence in a plurality of different types of        cells isolated from a subject when transduced into said cells.        83. The method of embodiment 82, comprising isolating said        population of cells.        84. The method of embodiment 83, comprising isolating said cells        via a non-invasive method.        85. The method of embodiment 84, comprising obtaining the cells        by saliva, sputum, sweat, urine, stool, semen, cervicovaginal        secretions, breast milk, rheum, tears, or cheek epithelium.        86. The method of embodiment 85, comprising isolating cells via        a minimally-invasive method.        87. The method of embodiment 86, comprising obtaining the cells        by venipuncture, thoracentesis, amniocentesis, or gastric        lavage.        88. The method of embodiment 87, comprising isolating said cells        by biopsy.        89. The method of any one of embodiments 82-88, wherein said        cells comprise cells found in blood or blood fractions, saliva,        urine, stool, cerebrospinal fluid, semen, vaginal secretions,        sputum, sweat, breast milk, synovial fluid, mucus (including        rheum), tears, bile, gastric fluid, interstitial fluid, biopsies        of tissues or epithelial cells that are naturally shed or        specifically collected from the body (such as cheek cell        scrapings), aqueous humor, amniotic fluid, pleural fluid or        breath exhalation.        90. The method of any one of embodiments 82-89, comprising        detecting said polypeptide or nucleic acid sequence.        91. The method of any one of embodiments 82-90, wherein said        disease is cancer, an autoimmune disease, or a neurodegenerative        disease.        92. The method of any one of embodiments 82-91, wherein said        promoter is a pan-cancer specific promoter.        93. A composition comprising a vector, wherein said vector        comprises a plurality of different promoters operably linked to        a plurality of barcode molecules, wherein each said promoter        drives expression of said plurality of barcode molecules in a        cell, and wherein levels of said plurality of barcode molecules        are indicative of a stage of a disease of said cell.        94. The composition of embodiment 93, wherein said stage of said        disease of said cell is diseased, non-diseased or an        intermediate state.        95. The composition of any one of embodiments 93-94, wherein        said disease is cancer, wherein said plurality of different        promoters comprises a first promoter activated in an early stage        of cancer.        96. The composition of any one of embodiments 93-95, wherein        said disease is cancer, wherein said plurality of different        promoters comprises a second promoter activated in an        intermediate stage of cancer.        97. The composition of any one of embodiments 93-96, wherein        said disease is cancer, wherein said plurality of different        promoters comprises a third promoter activated in a late stage        of cancer.        98. The composition of any one of embodiments 93-97, wherein        said vector is a non-viral vector.        99. The composition of any one of embodiments 93-98, wherein        said non-viral vector is a minicircle vector.        100. The composition of any one of embodiments 93-97, wherein        said vector is a nanoplasmid vector.        101. The composition of any one of embodiments 93-99, wherein        when said plurality of barcode molecule comprises a plurality of        polypeptide, each of said polypeptides is detectable by        non-invasive imaging.        102. The composition of embodiment 101, wherein non-invasive        imaging comprises photoacoustic, MRI, or PET imaging.        103. The composition of any one of embodiments 101, wherein each        of said polypeptides is detectable in a bodily sample.        104. The composition of embodiment 103, wherein said bodily        sample is blood or blood fractions, saliva, urine, stool,        cerebrospinal fluid, semen, vaginal secretions, sputum, sweat,        breast milk, synovial fluid, mucus (including rheum), tears,        bile, gastric fluid, interstitial fluid, biopsies of tissues or        epithelial cells that are naturally shed or specifically        collected from the body (such as cheek cell scrapings), aqueous        humor, amniotic fluid, pleural fluid or breath exhalation.        105. A method for detecting a stage of disease, comprising        administering to a subject a composition comprising a vector,        wherein said vector comprises:    -   a plurality of different promoters operably linked to a        plurality of different barcode molecules, wherein each said        promoter drives expression of said plurality of barcode        molecules in a cell,    -   wherein levels of individual barcode molecule are indicative of        a stage of a disease of said cell.        106. The method of embodiment 105, wherein said stage of said        disease of said cell is diseased, non-diseased or an        intermediate state.        107. The method of embodiment 105 or 106, said method determines        nodules, masses of tissue or lesions in said subject as        pre-cancerous, benign, dysplastic, or metastatic in nature.        108. The method of any one of embodiments 105-107, wherein said        disease is cancer, wherein said plurality of different promoters        comprises a first promoter activated in activated in an early        stage of cancer.        109. The method of any one of embodiments 105-108, wherein said        disease is cancer, wherein said plurality of different promoters        comprises a second promoter activated in an intermediate stage        of cancer.        110. The method of any one of embodiments 105-109, wherein said        disease is cancer, wherein said plurality of different promoters        comprises a third promoter activated in a late stage of cancer.        111. The method of any one of embodiments 105-110, wherein said        vector is a non-viral vector.        112. The method of any one of embodiments 105-111, wherein said        non-viral vector is a minicircle vector.        113. The method of any one of embodiments 105-110, wherein said        vector is a nanoplasmid vector.        114. The method of any one of embodiments 105-112, wherein when        said plurality of barcode molecules comprises a plurality of        polypeptides, the method further comprises detecting at least        one of said plurality of polypeptides by non-invasive imaging.        115. The method of embodiment 114, wherein non-invasive imaging        comprises photoacoustic, MRI, SPECT, or PET imaging.        116. The method of any one of embodiments 105-115, comprising at        detecting at least one of said plurality of barcode molecules in        a bodily sample.        117. The method of embodiment 116, wherein said bodily sample is        found in blood or blood fractions, saliva, urine, stool,        cerebrospinal fluid, semen, vaginal secretions, sputum, sweat,        breast milk, synovial fluid, mucus (including rheum), tears,        bile, gastric fluid, interstitial fluid, biopsies of tissues or        epithelial cells that are naturally shed or specifically        collected from the body (such as cheek cell scrapings), aqueous        humor, amniotic fluid, pleural fluid or breath exhalation.        118. The method of any one of embodiments 105-117, wherein said        composition is administered intravenously, subcutaneously,        intraventricularly, intrathecally, intracerebroventricularly,        transdermally, intramuscularly, orally, by inhalation, nasally,        rectally, intratumorally, proxi-tumorally, or into a lymph node        in said subject.        119. A composition comprising an engineered nucleic acid        encoding an expressible reporter gene that exhibits about 10% or        less expression in normal cells versus diseased cells when        compared to a recombinant nucleic acid comprising a reporter        gene comprising a nucleic acid sequence of SEQ ID NO: 1 or SEQ        ID NO: 2.        120. The composition of embodiment 119, wherein said engineered        nucleic acid comprises a pan-tumor specific promoter.        121. The composition of embodiment 120, wherein said pan-tumor        specific promoter comprises a transcriptional response element.        122. The composition of embodiment 121, wherein said        transcriptional response element comprises a modified p53        response element.        123. The composition of embodiment 122, wherein a modification        within said modified p53 response element results in decreased        promoter activity in normal cells relative to diseased cells.        124. The composition of embodiment 122 or 123, wherein a        modification within said modified p53 response element results        in increased promoter activity in diseased cells relative to        normal cells.        125. The composition of any one of embodiments 119-124, wherein        said disease is cancer.        126. A method comprising administering to a subject a        composition according to any one of embodiments 119-125.        127. The method of embodiment 126, wherein said subject is        suspected of having cancer.        128. The method of embodiment 125 or 126, wherein said        composition is administered intravenously, subcutaneously,        intraventricularly, intrathecally, intracerebroventricularly,        transdermally, intramuscularly, orally, by inhalation, nasally,        rectally, intratumorally, proxi-tumorally, or into a lymph node        in said subject.        129. A composition that exhibits about 10% or less expression in        normal cells versus diseased cells and comprises a recombinant        nucleic acid comprising a nucleic acid sequence encoding a        reporter gene that includes one or more miRNA binding sequences        in a 3′ untranslated region of said reporter gene.        130. The composition of embodiment 129, wherein binding of an        miRNA expressed in said diseased cells to at least one of the        one or more miRNA binding sequences results in reduced        translation of said reporter gene or reduction of a half-life of        an mRNA encoding said reporter gene.        131. The composition of embodiment 129 or 130, wherein said        diseased cell is a cancer cell.        132. The composition of embodiment 131, wherein said reporter        gene exhibits increased expression in said cancer cell due to        downregulation of at least one miRNA expressed in said cancer        cell.        133. The composition of any one of embodiments 129-132, wherein        said composition comprises at least two miRNA binding sequences        in said 3′ untranslated region of said reporter gene, wherein        two miRNA binding sequences have a substantially identical        nucleotide sequence capable of binding to a same miRNA.        134. The composition of any one of embodiments 129-133, wherein        said composition comprises at least two miRNA binding sequences        in said 3′ untranslated region of said reporter gene, wherein        said at least two miRNA binding sequences have different        nucleotide sequences, each of said different nucleotide        sequences capable of binding to different miRNAs.        135. The composition of any one of embodiments 129-134, wherein        said recombinant nucleic acid comprises DNA.        136. The composition of any one of embodiments 129-135, wherein        said recombinant nucleic acid is a synthetic or in        vitro-transcribed mRNA.        137. The composition of any one of embodiments 129-136, wherein        said one or more miRNA binding sequences comprise at least one        miR-15, miR-16, let-7, miR-122or miR-34 binding sequence.        138. A method of detecting a diseased cell, comprising        administering to a subject a composition according to any one of        embodiments 129-137.        139. The method of embodiment 138, comprising detecting said        reporter gene.        140. The method of embodiment 138, wherein said subject is        suspected of having cancer.        141. The method of any one of embodiments 138-140, wherein said        composition is administered intravenously, subcutaneously,        intraventricularly, intrathecally, intracerebroventricularly,        transdermally, intramuscularly, orally by inhalation, nasally,        rectally, intratumorally, proxi-tumorally, or into a lymph node        in said subject.        142. A composition exhibiting significantly longer expression of        synthetic biomarker versus plasmid DNA, minicircle DNA, or        nanoplasmid DNA comprising a linear vector comprising a        double-stranded nucleic acid comprising a promoter operatively        linked to a DNA sequence encoding a synthetic biomarker, wherein        a forward and a reverse strand of said double-stranded nucleic        acid are covalently linked on each of their terminal ends,        wherein said promoter induces expression of said synthetic        biomarker in a diseased cell preferentially over expression of        said synthetic biomarker in a non-diseased cell such that a        relative concentration of said synthetic biomarker expressed in        said diseased cell over said non-diseased cell is greater than        1.0.        143. The composition of embodiment 142, wherein said linear        vector comprises inverted terminal repeats (ITRs) flanking said        promoter operatively linked to said DNA sequence encoding said        synthetic biomarker, wherein said ITRs are derived from an        Adeno-Associated Virus (AAV).        144. The composition of embodiment 143, wherein said AAV is        AAV2.        145. The composition of any one of embodiments 142-144, wherein        said promoter drives expression of said synthetic biomarker        selectively in a plurality of diseased cells in a subject.        146. The composition of embodiment 145, wherein said promoter is        a Survivin promoter (BIRC5), a CXCR4 promoter, an ATP binding        cassette subfamily C member 4 (ABCC4) promoter, an anterior        gradient 2, protein disulphide isomerase family member (AGR2)        promoter, activation induced cytidine deaminase (AICDA)        promoter, an UDP-GlcNAc:betaGal        beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a        cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5        (CEACAM5) promoter, a centromere protein F (CENPF) promoter, a        centrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3)        promoter, a claudin 4 (CLDN4) promoter, a collagen type XI alpha        1 chain (COL11A1) promoter, a collagen type I alpha 1 chain        (COL1A1) promoter, a cystatin SN (CST1) promoter, a denticleless        E3 ubiquitin protein ligase homolog (DTL) promoter, a family        with sequence similarity 111 member B (FAM111B) promoter, a        forkhead box A1 (FOXA1) promoter, a kinesin family member 20A        (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitotic        spindle positioning (MISP) promoter, a matrix metallopeptidase 1        (MMP1) promoter, a matrix metallopeptidase 12 (MMP12) promoter,        a matrix metallopeptidase 13 (MMP13) promoter, a mesothelin        (MSLN) promoter, a cell surface associated mucin 1 (MUC1)        promoter, a phospholipase A2 group IID (PLA2G2D) promoter, a        regulator of G protein signaling 13 (RGS13) promoter, a        secretoglobin family 2A member 1 (SCGB2A1) promoter,        topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD)        promoter, a ubiquitin conjugating enzyme E2 C (UBE2C), a USH1        protein network component harmonin (USH1C), a V-set domain        containing T cell activation inhibitor 1 (VTCN1) promoter, a        Hexokinase type II promoter, a TRPM4 promoter, a stromelysin 3        promoter, a surfactant protein A promoter, a secretory        leukoprotease inhibitor promoter, a tyrosinase promoter, a        stress-inducible grp78/BiP promoter, an interleukin-10 promoter,        an α-B-crystallin/heat shock protein 27 promoter, an epidermal        growth factor receptor promoter, a mucin-like glycoprotein        promoter, an mts1 promoter, an NSE promoter, a somatostatin        receptor promoter, a c-erbB-3 promoter, a c-erbB-2 promoter, a        c-erbB4 promoter, a thyroglobulin promoter, an α-fetoprotein        promoter, a villin promoter, an albumin promoter, a glycoprotein        A33 promoter, the B cell-specific Moloney leukemia virus        insertion site 1 promoter, a cyclooxygenase-2 promoter, a        fibroblast growth factor promoter; a human epidermal growth        factor receptor 2, a human telomerase reverse transcriptase        promoter; a kinase domain insert containing receptor promoter; a        rad51 recombinase promoter; TTF-1, an urokinase-type plasminogen        activator receptor promoter, a ubiquitin conjugating enzyme E2 T        (UBE2T) promoter, a checkpoint kinase 1 (CHEK1) promoter, an        epithelial cell transforming 2 promoter (ECT2), a BCL2-like 12        (BCL2L12) promoter, a centromere protein I (CENPI) promoter, an        E2F transcription factor 1 (E2F1) promoter, a flavin adenine        dinucleotide synthetase 1 (FLAD1) promoter, a protein        phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an        ubiquitin conjugating enzyme E2 S (UBE2S) promoter, an aurora        kinase A and ninein interacting protein (AUNIP) promoter, a cell        division cycle 6 (CDC6) promoter, a centromere protein L (CENPL)        promoter, a DNA replication helicase/nuclease 2 (DNA2) promoter,        a DSN1 homolog, MIS12 kinetochore complex component (DSN1)        promoter, a deoxythymidylate kinase (DTYMK) promoter, a G        protein regulated inducer of neurite outgrowth 1 (GPRIN1)        promoter, a mitochondrial fission regulator 2 (MTFR2) promoter,        a RAD51 associated protein 1 (RAD51AP1) promoter, a small        nuclear ribonucleoprotein polypeptide A′ (SNRPA1) promoter, an        ATPase family, AAA domain containing 2 (ATAD2) promoter, a BUB1        mitotic checkpoint serine/threonine kinase (BUB1) promoter, a        calcyclin binding protein (CACYBP) promoter, a cell division        cycle associated 3 (CDCA3) promoter, a centromere protein O        (CENPO) promoter, a flap structure-specific endonuclease 1        (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, a cell        proliferation regulating inhibitor of protein phosphatase 2A        (KIAA1524) promoter, a kinesin family member 2C (KIF2C)        promoter, a karyopherin subunit alpha 2 (KPNA2) promoter, a MYB        proto-oncogene like 2 (MYBL2) promoter, a NIMA related kinase 2        (NEK2) promoter, a RAN binding protein 1 (RANBP1) promoter, a        small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB)        promoter, a SPC24/NDC80 kinetochore complex component (SPC24)        promoter, a transforming acidic coiled-coil containing protein 3        (TACC3) promoter, a TBC1 domain family member 31 (TBC1D31)        promoter, a thymidine kinase 1 (TK1) promoter, a zinc finger        protein 695 (ZNF695) promoter, an aurora kinase A (AURKA)        promoter, a BLM RecQ like helicase (BLM) promoter, a chromosome        17 open reading frame 53 (C17orf53) promoter, a chromobox 3        (CBX30) promoter, a cyclin B1 (CCNB1) promoter, a cyclin E1        (CCNE1) promoter, a cyclin F (CCNF), a cell division cycle 20        (CDC20) promoter, a cell division cycle 45 (CDC45) promoter, a        cell division cycle associated 5 (CDCA5) promoter, a cyclin        dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF        LAG seven-pass G-type receptor 3 (CELSR3) promoter, a centromere        protein A (CENPA) promoter, a centrosomal protein 72 (CEP72)        promoter, a CDC28 protein kinase regulatory subunit 2 (CKS2)        promoter, a collagen type X alpha 1 chain (COL10A1) promoter, a        chromosome segregation 1 like (CSE1L) promoter, a DBF4 zinc        finger promoter, a GINS complex subunit 1 (GINS1) promoter, a G        protein-coupled receptor 19 (GPR19) promoter, a kinesin family        member 18A (KIF18A) promoter, a kinesin family member 4A (KIF4A)        promoter, a kinesin family member C1 (KIFC1) promoter, a        minichromosome maintenance 10 replication initiation factor        (MCM10) promoter, a minichromosome maintenance complex component        2 (MCM2) promoter, a minichromosome maintenance complex        component 7 (MCM7) promoter, a MRG domain binding protein        (MRGBP) promoter, a methylenetetrahydrofolate dehydrogenase        (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase        (MTHFD2) promoter, a non-SMC condensin I complex subunit H        (NCAPH) promoter, a NDC80, kinetochore complex component (NDC80)        promoter, a nudix hydrolase 1 (NUDT1) promoter, a ribonuclease        H2 subunit A (RNASEH2A) promoter, a RuvB like AAA ATPase 1        (RUVBL1) promoter, a serologically defined breast cancer antigen        NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated        1 (SHCBP1) promoter, a small nuclear ribonucleoprotein        polypeptide G (SNRPG) promoter, a timeless circadian regulator        promoter, a thyroid hormone receptor interactor 13 (TRIP13)        promoter, a trophinin associated protein (TROAP) promoter, a        ubiquitin conjugating enzyme E2 C (UBE2C) promoter, a WD repeat        and HMG-box DNA binding protein 1 (WDHD1) promoter, an alpha        fetoprotein (AFP) promoter, a fragment thereof, or any        combination thereof.        147. The composition of any one of embodiments 142-146, wherein        said an MRI reporter, a PET reporter, a SPECT reporter, a        photoacoustic reporter, a bioluminescent reporter, a fluorescent        reporter, chemiluminescent reporter, luminescence reporter,        colorimetric reporter, a quantifiable nucleic acid biomarker and        combinations thereof and any combination thereof.        148. The composition of embodiment 147, wherein detection for        said biomarker determines a location of the diseased cell.        149. A method of identifying a diseased cell, comprising        administering to a subject a composition according to any one of        embodiments 142-148, and detecting said synthetic biomarker,        wherein said synthetic biomarker is expressed in a diseased cell        preferentially over expression of said synthetic biomarker in        non-diseased cells in said subject such that a relative        concentration of said synthetic biomarker expressed in said        diseased cell over said non-diseased cells is greater than 1.0.        150. The method of embodiment 149, wherein said diseased cell is        a cancer cell.        151. The method of any one of embodiments 149-150, wherein        detecting said synthetic biomarker identifies said diseased cell        with an accuracy of at least 90%.        152. The method of any one of embodiments 149-151, comprising        detecting said synthetic biomarker using a non-invasive imaging        method performed on said subject.        153. The method of any one of embodiments, wherein non-invasive        imaging comprises photoacoustic, MRI, SPECT, or PET imaging.        154. The method of any one of embodiments 149-153, comprising        detecting said synthetic biomarker in a bodily sample from said        subject.        155. The method of embodiment 154, wherein said bodily sample is        found in blood or blood fractions, saliva, urine, stool,        cerebrospinal fluid, semen, vaginal secretions, sputum, sweat,        breast milk, synovial fluid, mucus (including rheum), tears,        bile, gastric fluid, interstitial fluid, biopsies of tissues or        epithelial cells that are naturally shed or specifically        collected from the body (such as cheek cell scrapings), aqueous        humor, amniotic fluid, pleural fluid or breath exhalation.        156. The method of embodiment 154, wherein the sample is a        biopsy sample.        157. The method of embodiment 152 or 153, wherein detection of        said biomarker determines a location of the diseased cell.        158. The method of any one of embodiments 149-157, wherein said        subject is suspected of having cancer.        159. The method of any one of embodiments 149-158, wherein said        composition is administered intravenously, subcutaneously,        intraventricularly, intrathecally, intracerebroventricularly,        transdermally, intramuscularly, orally, by inhalation, nasally,        rectally, intratumorally, proxi-tumorally, or into a lymph node        in said subject.        160. A composition exhibiting significantly longer expression of        synthetic biomarker versus plasmid DNA or minicircle DNA        comprising a linear vector comprising a double-stranded nucleic        acid comprising a promoter operatively linked to a DNA sequence        encoding a therapeutically effective agent, wherein a forward        and a reverse strand of said double-stranded nucleic acid are        covalently linked on each of their terminal ends, wherein said        promoter induces expression of said therapeutically effective        agent in a diseased cell preferentially over expression of said        synthetic biomarker in a non-diseased cell such that a relative        concentration of said therapeutically effective agent expressed        in said diseased cell over said non-diseased cell is greater        than 1.0.        161. The composition of embodiment 160, wherein said linear        vector comprises inverted terminal repeats (ITRs) flanking said        promoter operatively linked to said DNA sequence encoding said        therapeutically effective agent, wherein said ITRs are derived        from an Adeno-Associated Virus (AAV).        162. The composition of embodiment 160 or 161, wherein said AAV        is AAV2.        163. The composition of any one of embodiments 160-162, wherein        said promoter drives expression of said therapeutically        effective agent selectively in a plurality of diseased cells in        a subject.        164. The composition of embodiment 163, wherein said promoter is        a Survivin promoter (BIRC5), a CXCR4 promoter, an ATP binding        cassette subfamily C member 4 (ABCC4) promoter, an anterior        gradient 2, protein disulphide isomerase family member (AGR2)        promoter, activation induced cytidine deaminase (AICDA)        promoter, an UDP-GlcNAc:betaGal        beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a        cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5        (CEACAM5) promoter, a centromere protein F (CENPF) promoter, a        centrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3)        promoter, a claudin 4 (CLDN4) promoter, a collagen type XI alpha        1 chain (COL11A1) promoter, a collagen type I alpha 1 chain        (COL1A1) promoter, a cystatin SN (CST1) promoter, a denticleless        E3 ubiquitin protein ligase homolog (DTL) promoter, a family        with sequence similarity 111 member B (FAM111B) promoter, a        forkhead box A1 (FOXA1) promoter, a kinesin family member 20A        (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitotic        spindle positioning (MISP) promoter, a matrix metallopeptidase 1        (MMP1) promoter, a matrix metallopeptidase 12 (MMP12) promoter,        a matrix metallopeptidase 13 (MMP13) promoter, a mesothelin        (MSLN) promoter, a cell surface associated mucin 1 (MUC1)        promoter, a phospholipase A2 group IID (PLA2G2D) promoter, a        regulator of G protein signaling 13 (RGS13) promoter, a        secretoglobin family 2A member 1 (SCGB2A1) promoter,        topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD)        promoter, a ubiquitin conjugating enzyme E2 C (UBE2C), a USH1        protein network component harmonin (USH1C), a V-set domain        containing T cell activation inhibitor 1 (VTCN1) promoter, a        Hexokinase type II promoter, a TRPM4 promoter, a stromelysin 3        promoter, a surfactant protein A promoter, a secretory        leukoprotease inhibitor promoter, a tyrosinase promoter, a        stress-inducible grp78/BiP promoter, an interleukin-10 promoter,        an α-B-crystallin/heat shock protein 27 promoter, an epidermal        growth factor receptor promoter, a mucin-like glycoprotein        promoter, an mts1 promoter, an NSE promoter, a somatostatin        receptor promoter, a c-erbB-3 promoter, a c-erbB-2 promoter, a        c-erbB4 promoter, a thyroglobulin promoter, an α-fetoprotein        promoter, a villin promoter, an albumin promoter, a glycoprotein        A33 promoter, the B cell-specific Moloney leukemia virus        insertion site 1 promoter, a cyclooxygenase-2 promoter, a        fibroblast growth factor promoter; a human epidermal growth        factor receptor 2, a human telomerase reverse transcriptase        promoter; a kinase domain insert containing receptor promoter; a        rad51 recombinase promoter; TTF-1, an urokinase-type plasminogen        activator receptor promoter, a ubiquitin conjugating enzyme E2 T        (UBE2T) promoter, a checkpoint kinase 1 (CHEK1) promoter, an        epithelial cell transforming 2 promoter (ECT2), a BCL2-like 12        (BCL2L12) promoter, a centromere protein I (CENPI) promoter, an        E2F transcription factor 1 (E2F1) promoter, a flavin adenine        dinucleotide synthetase 1 (FLAD1) promoter, a protein        phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an        ubiquitin conjugating enzyme E2 S (UBE2S) promoter, an aurora        kinase A and ninein interacting protein (AUNIP) promoter, a cell        division cycle 6 (CDC6) promoter, a centromere protein L (CENPL)        promoter, a DNA replication helicase/nuclease 2 (DNA2) promoter,        a DSN1 homolog, MIS12 kinetochore complex component (DSN1)        promoter, a deoxythymidylate kinase (DTYMK) promoter, a G        protein regulated inducer of neurite outgrowth 1 (GPRIN1)        promoter, a mitochondrial fission regulator 2 (MTFR2) promoter,        a RAD51 associated protein 1 (RAD51AP1) promoter, a small        nuclear ribonucleoprotein polypeptide A′ (SNRPA1) promoter, an        ATPase family, AAA domain containing 2 (ATAD2) promoter, a BUB1        mitotic checkpoint serine/threonine kinase (BUB1) promoter, a        calcyclin binding protein (CACYBP) promoter, a cell division        cycle associated 3 (CDCA3) promoter, a centromere protein O        (CENPO) promoter, a flap structure-specific endonuclease 1        (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, a cell        proliferation regulating inhibitor of protein phosphatase 2A        (KIAA1524) promoter, a kinesin family member 2C (KIF2C)        promoter, a karyopherin subunit alpha 2 (KPNA2) promoter, a MYB        proto-oncogene like 2 (MYBL2) promoter, a NIMA related kinase 2        (NEK2) promoter, a RAN binding protein 1 (RANBP1) promoter, a        small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB)        promoter, a SPC24/NDC80 kinetochore complex component (SPC24)        promoter, a transforming acidic coiled-coil containing protein 3        (TACC3) promoter, a TBC1 domain family member 31 (TBC1D31)        promoter, a thymidine kinase 1 (TK1) promoter, a zinc finger        protein 695 (ZNF695) promoter, an aurora kinase A (AURKA)        promoter, a BLM RecQ like helicase (BLM) promoter, a chromosome        17 open reading frame 53 (C17orf53) promoter, a chromobox 3        (CBX30) promoter, a cyclin B1 (CCNB1) promoter, a cyclin E1        (CCNE1) promoter, a cyclin F (CCNF), a cell division cycle 20        (CDC20) promoter, a cell division cycle 45 (CDC45) promoter, a        cell division cycle associated 5 (CDCA5) promoter, a cyclin        dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF        LAG seven-pass G-type receptor 3 (CELSR3) promoter, a centromere        protein A (CENPA) promoter, a centrosomal protein 72 (CEP72)        promoter, a CDC28 protein kinase regulatory subunit 2 (CKS2)        promoter, a collagen type X alpha 1 chain (COL10A1) promoter, a        chromosome segregation 1 like (CSE1L) promoter, a DBF4 zinc        finger promoter, a GINS complex subunit 1 (GINS1) promoter, a G        protein-coupled receptor 19 (GPR19) promoter, a kinesin family        member 18A (KIF18A) promoter, a kinesin family member 4A (KIF4A)        promoter, a kinesin family member C1 (KIFC1) promoter, a        minichromosome maintenance 10 replication initiation factor        (MCM10) promoter, a minichromosome maintenance complex component        2 (MCM2) promoter, a minichromosome maintenance complex        component 7 (MCM7) promoter, a MRG domain binding protein        (MRGBP) promoter, a methylenetetrahydrofolate dehydrogenase        (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase        (MTHFD2) promoter, a non-SMC condensin I complex subunit H        (NCAPH) promoter, a NDC80, kinetochore complex component (NDC80)        promoter, a nudix hydrolase 1 (NUDT1) promoter, a ribonuclease        H2 subunit A (RNASEH2A) promoter, a RuvB like AAA ATPase 1        (RUVBL1) promoter, a serologically defined breast cancer antigen        NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated        1 (SHCBP1) promoter, a small nuclear ribonucleoprotein        polypeptide G (SNRPG) promoter, a timeless circadian regulator        promoter, a thyroid hormone receptor interactor 13 (TRIP13)        promoter, a trophinin associated protein (TROAP) promoter, a        ubiquitin conjugating enzyme E2 C (UBE2C) promoter, a WD repeat        and HMG-box DNA binding protein 1 (WDHD1) promoter, an alpha        fetoprotein (AFP) promoter, a fragment thereof, or any        combination thereof.        165. The composition of any one of embodiments 160-164, wherein        said therapeutically effective agent is selected from the group        consisting of a therapeutically effective polypeptide, small        activating RNA (saRNA), microRNA (miRNA), small interfering RNA        (siRNA) or combinations of said therapeutically effective        polypeptide and said nucleic acids.        166. The composition of embodiment 165, wherein said        therapeutically effective agent is selected from the group        consisting of HSVtk, cytosine deaminase, DT diaphorase,        nitroreductase, guanine phosphoribosyl transferase, purine        nucleoside phosphorylase, thymidine phorphorylase,        carboxylesterase, folylpolyglutamyl synthetase, carboxypeptidase        A1, carboxypeptidase G2, cytochrome P-450.        167. A method of treating a diseased cell, comprising        administering to a subject a composition according to any one of        embodiments 160-166, and detecting said synthetic biomarker,        wherein said synthetic biomarker is expressed in a diseased cell        preferentially over expression of said synthetic biomarker in        non-diseased cells in said subject such that a relative        concentration of said synthetic biomarker expressed in said        diseased cell over said non-diseased cells is greater than 1.0.        168. The method of embodiment 167, wherein said diseased cell is        a cancer cell.        169. The method of any one of embodiments 167-168, wherein said        subject is suspected of having cancer.        170. The method of any one of embodiments 167-169 wherein said        composition is administered intravenously, subcutaneously,        intraventricularly, intrathecally, intracerebroventricularly,        transdermally, intramuscularly, orally, by inhalation, nasally,        rectally, intratumorally, proxi-tumorally, or into a lymph node        in said subject.        171. A composition comprising a non-viral vector expressing a        synthetic biomarker, wherein said synthetic biomarker exhibits        about 10% or less expression in normal organ cells versus        diseased cells.        172. The composition of embodiment 171, wherein said organ is        liver, kidney, spleen, or a combination thereof.        173. The composition of embodiment 171, wherein said non-viral        vector comprises a recombinant nucleic acid encoding a promoter        operably linked to a synthetic biomarker.        174. The composition of embodiment 173, wherein said promoter is        a Survivin promoter (BIRC5), a CXCR4 promoter, an ATP binding        cassette subfamily C member 4 (ABCC4) promoter, an anterior        gradient 2, protein disulphide isomerase family member (AGR2)        promoter, activation induced cytidine deaminase (AICDA)        promoter, an UDP-GlcNAc:betaGal        beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, a        cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5        (CEACAM5) promoter, a centromere protein F (CENPF) promoter, a        centrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3)        promoter, a claudin 4 (CLDN4) promoter, a collagen type XI alpha        1 chain (COL11A1) promoter, a collagen type I alpha 1 chain        (COL1A1) promoter, a cystatin SN (CST1) promoter, a denticleless        E3 ubiquitin protein ligase homolog (DTL) promoter, a family        with sequence similarity 111 member B (FAM111B) promoter, a        forkhead box A1 (FOXA1) promoter, a kinesin family member 20A        (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitotic        spindle positioning (MISP) promoter, a matrix metallopeptidase 1        (MMP1) promoter, a matrix metallopeptidase 12 (MMP12) promoter,        a matrix metallopeptidase 13 (MMP13) promoter, a mesothelin        (MSLN) promoter, a cell surface associated mucin 1 (MUC1)        promoter, a phospholipase A2 group IID (PLA2G2D) promoter, a        regulator of G protein signaling 13 (RGS13) promoter, a        secretoglobin family 2A member 1 (SCGB2A1) promoter,        topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD)        promoter, a ubiquitin conjugating enzyme E2 C (UBE2C), a USH1        protein network component harmonin (USH1C), a V-set domain        containing T cell activation inhibitor 1 (VTCN1) promoter, a        Hexokinase type II promoter, a TRPM4 promoter, a stromelysin 3        promoter, a surfactant protein A promoter, a secretory        leukoprotease inhibitor promoter, a tyrosinase promoter, a        stress-inducible grp78/BiP promoter, an interleukin-10 promoter,        an α-B-crystallin/heat shock protein 27 promoter, an epidermal        growth factor receptor promoter, a mucin-like glycoprotein        promoter, an mts1 promoter, an NSE promoter, a somatostatin        receptor promoter, a c-erbB-3 promoter, a c-erbB-2 promoter, a        c-erbB4 promoter, a thyroglobulin promoter, an α-fetoprotein        promoter, a villin promoter, an albumin promoter, a glycoprotein        A33 promoter, the B cell-specific Moloney leukemia virus        insertion site 1 promoter, a cyclooxygenase-2 promoter, a        fibroblast growth factor promoter; a human epidermal growth        factor receptor 2, a human telomerase reverse transcriptase        promoter; a kinase domain insert containing receptor promoter; a        rad51 recombinase promoter; TTF-1, an urokinase-type plasminogen        activator receptor promoter, a ubiquitin conjugating enzyme E2 T        (UBE2T) promoter, a checkpoint kinase 1 (CHEK1) promoter, an        epithelial cell transforming 2 promoter (ECT2), a BCL2-like 12        (BCL2L12) promoter, a centromere protein I (CENPI) promoter, an        E2F transcription factor 1 (E2F1) promoter, a flavin adenine        dinucleotide synthetase 1 (FLAD1) promoter, a protein        phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an        ubiquitin conjugating enzyme E2 S (UBE2S) promoter, an aurora        kinase A and ninein interacting protein (AUNIP) promoter, a cell        division cycle 6 (CDC6) promoter, a centromere protein L (CENPL)        promoter, a DNA replication helicase/nuclease 2 (DNA2) promoter,        a DSN1 homolog, MIS12 kinetochore complex component (DSN1)        promoter, a deoxythymidylate kinase (DTYMK) promoter, a G        protein regulated inducer of neurite outgrowth 1 (GPRIN1)        promoter, a mitochondrial fission regulator 2 (MTFR2) promoter,        a RAD51 associated protein 1 (RAD51AP1) promoter, a small        nuclear ribonucleoprotein polypeptide A′ (SNRPA1) promoter, an        ATPase family, AAA domain containing 2 (ATAD2) promoter, a BUB1        mitotic checkpoint serine/threonine kinase (BUB1) promoter, a        calcyclin binding protein (CACYBP) promoter, a cell division        cycle associated 3 (CDCA3) promoter, a centromere protein O        (CENPO) promoter, a flap structure-specific endonuclease 1        (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, a cell        proliferation regulating inhibitor of protein phosphatase 2A        (KIAA1524) promoter, a kinesin family member 2C (KIF2C)        promoter, a karyopherin subunit alpha 2 (KPNA2) promoter, a MYB        proto-oncogene like 2 (MYBL2) promoter, a NIMA related kinase 2        (NEK2) promoter, a RAN binding protein 1 (RANBP1) promoter, a        small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB)        promoter, a SPC24/NDC80 kinetochore complex component (SPC24)        promoter, a transforming acidic coiled-coil containing protein 3        (TACC3) promoter, a TBC1 domain family member 31 (TBC1D31)        promoter, a thymidine kinase 1 (TK1) promoter, a zinc finger        protein 695 (ZNF695) promoter, an aurora kinase A (AURKA)        promoter, a BLM RecQ like helicase (BLM) promoter, a chromosome        17 open reading frame 53 (C17orf53) promoter, a chromobox 3        (CBX30) promoter, a cyclin B1 (CCNB1) promoter, a cyclin E1        (CCNE1) promoter, a cyclin F (CCNF), a cell division cycle 20        (CDC20) promoter, a cell division cycle 45 (CDC45) promoter, a        cell division cycle associated 5 (CDCA5) promoter, a cyclin        dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF        LAG seven-pass G-type receptor 3 (CELSR3) promoter, a centromere        protein A (CENPA) promoter, a centrosomal protein 72 (CEP72)        promoter, a CDC28 protein kinase regulatory subunit 2 (CKS2)        promoter, a collagen type X alpha 1 chain (COL10A1) promoter, a        chromosome segregation 1 like (CSE1L) promoter, a DBF4 zinc        finger promoter, a GINS complex subunit 1 (GINS1) promoter, a G        protein-coupled receptor 19 (GPR19) promoter, a kinesin family        member 18A (KIF18A) promoter, a kinesin family member 4A (KIF4A)        promoter, a kinesin family member C1 (KIFC1) promoter, a        minichromosome maintenance 10 replication initiation factor        (MCM10) promoter, a minichromosome maintenance complex component        2 (MCM2) promoter, a minichromosome maintenance complex        component 7 (MCM7) promoter, a MRG domain binding protein        (MRGBP) promoter, a methylenetetrahydrofolate dehydrogenase        (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase        (MTHFD2) promoter, a non-SMC condensin I complex subunit H        (NCAPH) promoter, a NDC80, kinetochore complex component (NDC80)        promoter, a nudix hydrolase 1 (NUDT1) promoter, a ribonuclease        H2 subunit A (RNASEH2A) promoter, a RuvB like AAA ATPase 1        (RUVBL1) promoter, a serologically defined breast cancer antigen        NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated        1 (SHCBP1) promoter, a small nuclear ribonucleoprotein        polypeptide G (SNRPG) promoter, a timeless circadian regulator        promoter, a thyroid hormone receptor interactor 13 (TRIP13)        promoter, a trophinin associated protein (TROAP) promoter, a        ubiquitin conjugating enzyme E2 C (UBE2C) promoter, a WD repeat        and HMG-box DNA binding protein 1 (WDHD1) promoter, an alpha        fetoprotein (AFP) promoter, a fragment thereof, or any        combination thereof.        175. The composition of embodiment 173 or 174, wherein said        recombinant nucleic acid comprises regulatory elements that        silence or attenuate transcription or mRNA half-life of said        nucleic acid sequence in normal liver cells.        176. The composition of embodiment 175, wherein said regulatory        elements comprise one or more miRNA target sequences in a        transcribed, but an untranslated region, of said recombinant        nucleic acid.        177. The composition of embodiment 176, wherein the presence of        the one or more miRNA target sequences inhibits expression from        said recombinant nucleic acid sequence.        178. The composition of any one of embodiments 176-177, wherein        said one or more miRNA target sequences comprise at least one        miRNA target sequence for at least one tissue specific miRNA.        179. The composition of embodiment 178, wherein said at least        one tissue specific miRNA comprises at least one miRNA enriched        in normal hepatic tissues.        180. The composition of embodiment 179, wherein said at least        one miRNA enriched in normal hepatic tissues comprises miR-122,        miR-33, miR-33*, miR-223, miR-30c, miR-144, miR-148a, miR-24,        miR-29, or any combination thereof.        181. The composition of any one of embodiments 170-180, wherein        said composition further comprises a transfection agent.        182. The composition of embodiment 181, wherein said        transfection agent is a linear or branched polyethylenimine,        nanoparticle, lipophilic particle, solid nanoparticle, peptides,        micelles, dendrimers, polymeric compositions, hydrogels,        synthetic or naturally derived nanocell, exosomes, a virus-like        particle, or any combination thereof.        183. An engineered particle that mimics one or many functions of        a biological cell or macrophage including inducing the        expression of a biomarker in a diseased cell preferentially over        expression of said biomarker in non-diseased cells such that        said relative concentration ratio of said biomarker expressed in        said diseased cell over said non-diseased cells is greater than        1.0.        184. The engineered particle of embodiment 183, wherein said        engineered particle is an artificial cell, a minimal cell, or a        lipid-enclosed synthetic particle.        185. The engineered particle of embodiment 183 or 184, wherein        said engineered particle comprises biological membranes,        polymeric membranes, simple polymers, crosslinked proteins,        lipid membranes or polymer-lipid complexes formed in vitro or in        vivo with purified components into nanoparticles, liposomes,        polymersomes, exosomes, microvesicles, apoptotic blebs,        transport vesicles, synaptic vesicles, secretory vesicles, or        microcapsules.        186. The engineered particle of any one of embodiments 183-185,        wherein said engineered particle comprises a transmembrane        chimeric protein or a naturally occurring protein comprising an        extraparticle specific binding domain operably linked to an        intraparticle signaling domain, wherein said intraparticle        signaling domain is capable of activating at least one enzymatic        reaction within said engineered particle.        187. The engineered particle of embodiment 186, wherein said        engineered particle comprises a nucleic acid sequence encoding a        synthetic biomarker, wherein said at least one enzymatic        reaction results in the production of said synthetic biomarker        within said engineered particle.        188. The engineered particle of embodiment 186 or 187, wherein        said extraparticle specific binding domain comprises an scFv or        a Fab fragment.        189. The engineered particle of any one of embodiments 183-188,        wherein said engineered particle comprises cytoplasm and other        components isolated from intact cells        190. The engineered particle of any one of embodiments 183-173,        wherein said engineered particle comprises purified recombinant        macromolecular components or macromolecular components such as,        but not limited to, cellular proteins, DNA, synthetic gene        circuits, organelles, ATP, enzymes, NADP, transcription factors,        nucleotides, or cell-free transcription-translation extracts.        191. At least one vector, wherein said at least one vector        comprises:    -   a plurality of different promoters operably linked to a        plurality of different nucleic acid sequences, wherein said        promoters drive expression of said plurality of nucleic acid        sequences in a cell to yield a plurality of polypeptides or        nucleic acid biomarker sequences,    -   wherein said promoters induce expression of said plurality of        polypeptides or nucleic acid biomarker sequences in a diseased        cell preferentially over expression of said plurality of        polypeptides or nucleic acid biomarker sequences in non-diseased        cells in a subject such that a relative ratio of said plurality        of polypeptides or nucleic acid biomarker sequences expressed in        said diseased cell over said non-diseased cells is greater than        1.0.        192. The vector of embodiment 191, wherein each of said        promoters induce expression of said plurality of polypeptides or        nucleic acid biomarker sequences in a diseased cell        preferentially over expression of said plurality of polypeptides        or nucleic acid biomarker sequences in non-diseased cells in        said subject such that a relative ratio of said plurality of        polypeptides or nucleic acid biomarker sequences expressed in        said diseased cell over said non-diseased cells is greater than        1.0.        193. The vector of embodiment 191 or 192, wherein said plurality        of different promoters comprises at least 2, at least 3, at        least 4, or at least 5 promoters.        194. The vector of any one of embodiments 191-193, wherein said        plurality of different promoters comprises at most 12, at most        13, at most 14, or at most 15 promoters.        195. The vector of any one of embodiments 191-194, wherein at        least two of said plurality of different promoters are selected        from Table 1.        196. The vector of any one of embodiments 191-195, wherein said        vector is a plasmid, nanoplasmid, minicircle, recombinant viral        vector, or CELiD.        197. The vector of embodiment 196, wherein said minicircle is a        self-replicating minicircle.        198. The vector of embodiment 197, wherein said self-replicating        minicircle comprises an S/MAR element.        199. The vector of any one of embodiments 191-198, wherein said        plurality of polypeptides or nucleic acid biomarker sequences        comprises at least one reporter polypeptide selected from the        group consisting of a photoacoustic reporter, a bioluminescent        reporter, an autofluorescent reporter, a chemiluminescent        reporter, a luminescent reporter, or a colorimetric reporter, or        any combination thereof.        200. The vector of any one of embodiments 191-199, wherein said        plurality of polypeptides or nucleic acid biomarker sequences        comprises at least one polypeptide with an N-terminal secretion        signal sequence.        201. The vector of any one of embodiments 191-200, wherein said        plurality of polypeptides or nucleic acid biomarker sequences        comprises at least one nucleic acid biomarker sequence selected        from the group consisting of a ribozyme, a self-splicing intron,        a microRNA, a RNA aptamer, or another type of quantifiable RNA        biomarker.        202. The vector of any one of embodiments 191-201, wherein said        disease is cancer, an autoimmune disease, or a neurodegenerative        disease.        203. A method for detecting a disease in a subject, comprising:    -   administering to a subject a composition comprising the vector        according to any one of embodiments 191-201;    -   detecting said plurality of polypeptides or nucleic acid        biomarker sequences to obtain an expression profile; and    -   detecting said diseased cell based said expression profile,        thereby detecting said disease.        204. The method of embodiment 203, wherein (b) comprises        detecting said plurality of polypeptides or nucleic acid        biomarker sequences from a sample from said subject.        205. The method of embodiment 204, wherein said sample is a        bodily fluid from said subject.        206. The method of embodiment 205, wherein said biological        sample is a blood or blood-based sample from said subject.        207. The method of any one of embodiments 203-206, wherein said        plurality of polypeptides or nucleic acid biomarker sequences        comprises at least one nucleic acid biomarker sequence,        wherein (b) comprises detecting said at least one nucleic acid        biomarker sequence by quantitative PCR, sequencing, or a        hybridization-based technique.        208. The method of any one of embodiments 203-207, wherein said        plurality of polypeptides or nucleic acid biomarker sequences        comprises at least one reporter polypeptide, comprising        detecting said at least one reporter polypeptide by        photoacoustic assay, bioluminescence assay, fluorescence assay,        chemiluminescent assay, colorimetric assay, or any combination        thereof.        209. The method of any one of embodiments 203-208, wherein (c)        comprises applying a machine learning or classifier algorithm to        said expression profile, wherein said machine learning or        classifier algorithm is configured to distinguish between an        expression profile indicative of a diseased cell from an        expression profile indicative of a non-diseased cell.        210. The method of any one of embodiments 203-209, wherein said        composition is administered intravenously, subcutaneously,        intraventricularly, intrathecally, intracerebroventricularly,        transdermally, intramuscularly, orally, by inhalation, nasally,        rectally, intratumorally, proxi-tumorally, or into a lymph node        in said subject.        211. The method of any one of embodiments 203-210, wherein said        composition further comprises a pharmaceutically acceptable        carrier.        212. The method of any one of embodiments 203-211, wherein said        composition further comprises a transfection agent.        213. A method for detecting a subject's disease or absence        thereof, comprising:    -   (a) contacting one or more cells of said subject with a genetic        construct ex-vivo, wherein:    -   said genetic construct comprises a disease-activated promoter        operably linked to a barcode molecule and said disease-activated        promoter drives expression of said barcode molecule in a cell        affected by said disease;    -   (b) quantifying an expression level of said barcode molecule;        and    -   (c) detecting said disease or absence thereof based on said        expression level.        214. A method for generating a profile of a subject's disease,        comprising:    -   (a) contacting one or more cells of said subject with a        plurality of genetic constructs, wherein:    -   said plurality of genetic constructs comprises a plurality of        disease-activated promoters respectively operably linked to a        plurality of barcode molecules and said disease-activated        promoter drives expression of said corresponding barcode        molecule in a cell affected by said disease; and    -   (b) quantifying expression levels of said plurality of barcode        molecules to generate said profile.        215. The method of embodiment 214, wherein said contacting is        ex-vivo.        216. The method of embodiment 214, wherein said contacting is in        vivo.        217. The method of embodiment 213 or embodiment 214, further        comprising isolating said one or more cells from said subject.        218. The method of embodiment 213 or embodiment 214, wherein        said disease-activated promoter comprises a cancer-activated        promoter or said plurality of disease-activated promoters        comprise a plurality of cancer-specific promoters.        219. The method of embodiment 218, wherein the cancer-activated        promoter or plurality of cancer-activated promoters are        activated in Acute Myeloid Leukemia, Adrenocortical Carcinoma,        Bladder Urothelial Carcinoma, Breast Ductal Carcinoma, Breast        Lobular Carcinoma, Cervical Carcinoma, Cholangiocarcinoma,        Colorectal Adenocarcinoma, Esophageal Carcinoma, Gastric        Adenocarcinoma, Glioblastoma Multiforme, Head and Neck Squamous        Cell Carcinoma, Hepatocellular Carcinoma, Kidney Chromophobe        Carcinoma, Kidney Clear Cell Carcinoma, Kidney Papillary Cell        Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma, Lung        Squamous Cell Carcinoma, Mesothelioma, Ovarian Serous        Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Paraganglioma        & Pheochromocytoma, Prostate Adenocarcinoma, Sarcoma, Skin        Cutaneous Melanoma, Testicular Germ Cell Cancer, Thymoma,        Thyroid Papillary Carcinoma, Uterine Carcinosarcoma, Uterine        Corpus Endometrioid Carcinoma, Uveal Melanoma, lip melanoma,        spindle cell carcinoma, liposarcoma, nasal sarcoma, mammary        adenocarcinoma, insulinoma, osteosarcoma, mast cell tumors,        hemangiosarcoma, non-small cell lung carcinoma (NSCLC), marginal        lymphoma, malignant melanoma, or chronic lymphocytic leukemia.        220. The method of any one of embodiments 218-219, comprising a        plurality of cancer-activated promoters, wherein said plurality        of cancer-activated promoters comprises promoters activated in a        plurality of cancers within different tissue origins.        221. The method of any one of embodiments 220, wherein said        plurality of cancer-specific promoters are activated in a        plurality (e.g. two or more, three or more, four or more, five        or more, six or more, seven or more, eight or more, nine or        more, ten or more, eleven or more, twelve or more, thirteen or        more, fourteen or more, fifteen or more, sixteen or more,        seventeen or more, eighteen or more, nineteen or more, twenty or        more, twenty-five or more, or thirty or more) of different        tissue origins.        222. The method of any one of embodiments 220-221, wherein said        plurality of cancer-specific promoters comprise a first promoter        that produces a strong signal, a second promoter that produces a        low background signal, a third promoter that has a high        signal-to-background signal ratio, or any combinations thereof.        223. The method of embodiment 222, wherein said plurality of        cancer-specific promoters comprises at least one said first        promoter, at least one said second promoter, and at least one        said third promoter.        224. The method of embodiment 214, wherein said plurality of        disease-activated promoters comprise a plurality of        cancer-activated promoters that are selective for and activated        in a selected group of tissue origin.        225. The method of embodiment 214, wherein said plurality of        disease-activated promoters comprises a plurality of        cancer-activated promoters that are selective for and activated        in the same tissue origin.        226. The method of embodiment 214, wherein said plurality of        disease-activated promoters comprises a plurality of        cancer-activated promoters activated in a plurality of different        molecular subtypes of a cancer respectively within the same        tissue origin.        227. The method of embodiment 214, wherein said plurality of        disease-specific promoters comprise a plurality of        cancer-activated promoters activated in one molecular subtype of        a cancer within a tissue origin.        228. The method of embodiment 214, wherein said plurality of        genetic constructs comprises two or more different        cancer-activated promoters activated in one stage of a cancer        within a tissue origin.        229. The method of any of embodiments 213-228, comprising        contacting at least one cell with said plurality of genetic        constructs or said genetic construct.        230. The method of any of embodiments 213-229, wherein said        genetic construct or said plurality of genetic constructs        comprises a non-viral vector.        231. The method of embodiment 230, wherein said non-viral vector        is a nanoplasmid.        232. The method of any of embodiments 213-229, wherein said        genetic construct or said plurality of genetic constructs        comprises a replication-incompetent recombinant virion or an        isolated inverted terminal repeat (ITRs) derived therefrom.        233. The method of embodiment 232, wherein said virion is a        lentiviral, adeno-associated viral, adenoviral, or        gamma-retroviral virion.        234. The method of embodiment 232, wherein said virion is        derived from a virus with primarily episomal genome maintenance        within infected cells.        235. The method of embodiment 233, wherein said        replication-incompetent virion is a recombinant adenovirus        vector.        236. The method of embodiment 235, wherein said AAV is serotype        1, 2, 3, 4, 5, 6, 8, 9, Ad5, Ad-RGD, or Ad-19a/64, or a        pseudotyped variant thereof.        237. The method of any one of embodiments 232-236, wherein said        replication-incompetent recombinant virion is combined with said        cells from said subject at a multiplicity of infection (MOI) of        0.001-10.        238. The method of any one of embodiments 213-237, wherein said        subject is human or canine.        239. The method of any one of embodiments 213-238, wherein said        subject has previously received surgical, chemotherapeutic,        radiological or immunotherapeutic treatment for cancer.        240. The method of any one of embodiments 213-239, wherein said        subject has at least one risk factor for cancer such as, having        Li-Fraumeni syndrome, lynch syndrome, familial adenomatous        polyposis, Von Hippel-Lindau disease, aplastic anemia,        myelodysplastic syndrome, Cowden syndrome, hereditary breast and        ovarian cancer syndrome (HBOC), or BRCA mutations; being a        current smoker, ex-smoker or exposed to heavy doses of second        hand smoke; exposure to carcinogens, excessive sunlight,        immunosuppressive agents, infectious agents such as hepatitis B        or C as well as human papilloma virus; or being obese.        241. The method of any one of embodiments 213-239, wherein said        subject has at least one symptom of cancer, such as a positive        signal on a mammogram or from a cancer screening diagnostic        test, disproportionate blood cell distribution, weight loss,        swollen lumps or glands, night sweats, blood in urine, blood in        stool, unexpected bleeding or discharge from body including        nipples, dizziness, blurred vision or loss of balance, diarrhea,        acute pain, low grade fever, constipation, loss of appetite,        nagging cough or hoarseness, or jaundice.        242. The method of any of embodiments 214-241, further        comprising (c) detecting said disease based on said profile.        243. The method of embodiment 242, further comprising processing        said profile comprising expression levels of said barcode        molecules corresponding to said plurality of disease-activated        promoters using a classifier to detect said disease or absence        thereof.        244. The method of embodiment 243, wherein detecting said        disease or absence thereof further comprises determining a        tissue origin.        245. The method of embodiment 213, comprising detecting said        disease or absence thereof with an AUC of at least 65%, 70%,        75%, 80%, 85%, 90%, or 95%.        246. The method of embodiment 213, comprising detecting said        disease or absence thereof with a sensitivity of at least 65%,        70%, 75%, 80%, 85%, 90%, or 95%.        247. The method of embodiment 213, comprising detecting said        disease or absence thereof with a specificity of at least 65%,        70%, 75%, 80%, 85%, 90%, or 95%.        248. The method of embodiment 242, further comprising,        subsequent to (c), (d) using one or more disease-activated        promoters corresponding to a disease detected in (c) to        repeat (a) and (b) to confirm said disease detected in (c)        and/or detect a subtype of said disease detected in (c).        249. The method of embodiment 248, further comprising        repeating (d) to confirm said disease detected in (c) and/or        detect a subtype of said disease detected in (c).        250. The method of any one of embodiments 213-249, wherein a        barcode molecule uniquely identifies a disease-specific promoter        of said genetic construct.        251. The method of any one of embodiments 213-249, wherein said        barcode molecule comprises a nucleotide sequence or a peptide        sequence.        252. The method of embodiment 251, wherein said barcode molecule        comprises a nucleotide sequence, wherein said nucleotide        sequence comprises a unique DNA or RNA.        253. The method of embodiment 252, wherein said barcode molecule        comprises RNA, wherein said RNA is a barcode processed from a        miRNA scaffold.        254. The method of embodiment 253, wherein said miRNA scaffold        comprises 5′, 3′, and loop regions derived said miRNA scaffold,        and stem regions comprising said barcode.        255. The method of embodiment 251, wherein said barcode molecule        comprises a peptide sequence, wherein said peptide sequence        comprises an enzyme reporter.        256. The method of embodiment 251, wherein said enzyme reporter        is a luciferase, an EGFP, a GFP, an RFP, horseradish peroxidase        (HRP), an alkaline phosphatase (AP), glucose oxidase (GO), or        beta galactosidase (BGAL), or any combination thereof.        257. The method of any one of embodiments 213-256, wherein said        barcode molecule is secretable or sheddable from said cells.        258. The method of embodiment 257, comprising quantifying an        expression level of said barcode or expression levels of said        barcodes in extracellular fluid in contact with said cells.        259. The method of embodiment 258, wherein said contact with        said cells is in vivo and said extracellular fluid comprises        blood or blood fractions, saliva, urine, stool, cerebrospinal        fluid, semen, vaginal secretions, sputum, sweat, breast milk,        synovial fluid, mucus (including rheum), tears, bile, gastric        fluid, interstitial fluid, aqueous humor, amniotic fluid, or        pleural fluid.        260. The method of embodiment 257 or embodiment 258, comprising        quantifying an expression level of said barcode or expression        levels of said barcodes in extracellular fluid at least 8 hours,        at least 12 hours, at least 24 hours, at least 36 hours, at        least 48 hours, at least 72 hours following said contact with        said cells.        261. The method of any one of embodiments 252-254, wherein said        quantifying comprises sequencing said barcode molecule or        plurality of barcode molecules.        262. The method of any one of embodiments 255-257, wherein said        quantifying comprises measuring an activity of said enzyme        reporter or reporters.        263. The method of embodiment 262, wherein said activity of said        enzyme reporter or reporters is measure by an immunochemistry        reaction, lateral flow electrophoresis, or mass        spectrophotometer.        264. The method of any of embodiments 214-265, further        comprising transmitting said profile on an intranet or the        internet.        265. The method of any of embodiments 213-264, further        comprising providing a therapeutic treatment based on said        detecting said disease or absence thereof or said profile.

1. A method comprising: (a) administering to a subject a composition,wherein said composition induces expression of a biomarker in a diseasedcell preferentially over expression of said biomarker in non-diseasedcells in said subject such that a relative ratio of said biomarkerexpressed in said diseased cell over said non-diseased cells is greaterthan 1.0; (b) detecting said biomarker; and (c) using said biomarkerdetected in (b) to determine that said subject has said diseased cell atan accuracy of at least 70%, wherein said promoter is selected from thegroup consisting of a Survivin promoter (BIRC5), a CXCR4 promoter, anATP binding cassette subfamily C member 4 (ABCC4) promoter, an anteriorgradient 2, protein disulphide isomerase family member (AGR2) promoter,activation induced cytidine deaminase (AICDA) promoter, anUDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3)promoter, a cadherin 3 (CDH3) promoter, a CEA cell adhesion molecule 5(CEACAM5) promoter, a centromere protein F (CENPF) promoter, acentrosomal protein 55 (CEP55) promoter, a claudin 3 (CLDN3) promoter, aclaudin 4 (CLDN4) promoter, a collagen type XI alpha 1 chain (COL11A1)promoter, a collagen type I alpha 1 chain (COL1A1) promoter, a cystatinSN (CST1) promoter, a denticleless E3 ubiquitin protein ligase homolog(DTL) promoter, a family with sequence similarity 111 member B (FAM111B)promoter, a forkhead box A1 (FOXA1) promoter, a kinesin family member20A (KIF20A), a laminin subunit gamma 2 (LAMC2) promoter, a mitoticspindle positioning (MISP) promoter, a matrix metallopeptidase 1 (MMP1)promoter, a matrix metallopeptidase 12 (MMP12) promoter, a matrixmetallopeptidase 13 (MMP13) promoter, a mesothelin (MSLN) promoter, acell surface associated mucin 1 (MUC1) promoter, a phospholipase A2group IID (PLA2G2D) promoter, a regulator of G protein signaling 13(RGS13) promoter, a secretoglobin family 2A member 1 (SCGB2A1) promoter,topoisomerase II alpha (TOP2A) promoter, a ubiquitin D (UBD) promoter, aubiquitin conjugating enzyme E2 C (UBE2C), a USH1 protein networkcomponent harmonin (USH1C), a V-set domain containing T cell activationinhibitor 1 (VTCN1) promoter, a Hexokinase type II promoter, a TRPM4promoter, a stromelysin 3 promoter, a surfactant protein A promoter, asecretory leukoprotease inhibitor promoter, a tyrosinase promoter, astress-inducible grp78/BiP promoter, an interleukin-10 promoter, anα-B-crystallin/heat shock protein 27 promoter, an epidermal growthfactor receptor promoter, a mucin-like glycoprotein promoter, an mts1promoter, an NSE promoter, a somatostatin receptor promoter, a c-erbB-3promoter, a c-erbB-2 promoter, a c-erbB4 promoter, a thyroglobulinpromoter, an α-fetoprotein promoter, a villin promoter, an albuminpromoter, a glycoprotein A33 promoter, the B cell-specific Moloneyleukemia virus insertion site 1 promoter, a cyclooxygenase-2 promoter, afibroblast growth factor promoter; a human epidermal growth factorreceptor 2, a human telomerase reverse transcriptase promoter; a kinasedomain insert containing receptor promoter; a rad51 recombinasepromoter; TTF-1, an urokinase-type plasminogen activator receptorpromoter, a ubiquitin conjugating enzyme E2 T (UBE2T) promoter, acheckpoint kinase 1 (CHEK1) promoter, an epithelial cell transforming 2promoter (ECT2), a BCL2-like 12 (BCL2L12) promoter, a centromere proteinI (CENPI) promoter, an E2F transcription factor 1 (E2F1) promoter, aflavin adenine dinucleotide synthetase 1 (FLAD1) promoter, a proteinphosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) promoter, an ubiquitinconjugating enzyme E2 S (UBE2S) promoter, an aurora kinase A and nineininteracting protein (AUNIP) promoter, a cell division cycle 6 (CDC6)promoter, a centromere protein L (CENPL) promoter, a DNA replicationhelicase/nuclease 2 (DNA2) promoter, a DSN1 homolog, MIS12 kinetochorecomplex component (DSN1) promoter, a deoxythymidylate kinase (DTYMK)promoter, a G protein regulated inducer of neurite outgrowth 1 (GPRIN1)promoter, a mitochondrial fission regulator 2 (MTFR2) promoter, a RAD51associated protein 1 (RAD51AP1) promoter, a small nuclearribonucleoprotein polypeptide A′ (SNRPA1) promoter, an ATPase family,AAA domain containing 2 (ATAD2) promoter, a BUB1 mitotic checkpointserine/threonine kinase (BUB1) promoter, a calcyclin binding protein(CACYBP) promoter, a cell division cycle associated 3 (CDCA3) promoter,a centromere protein O (CENPO) promoter, a flap structure-specificendonuclease 1 (FEN1) promoter, a forkhead box M1 (FOXM1) promoter, acell proliferation regulating inhibitor of protein phosphatase 2A(KIAA1524) promoter, a kinesin family member 2C (KIF2C) promoter, akaryopherin subunit alpha 2 (KPNA2) promoter, a MYB proto-oncogene like2 (MYBL2) promoter, a NIMA related kinase 2 (NEK2) promoter, a RANbinding protein 1 (RANBP1) promoter, a small nuclear ribonucleoproteinpolypeptides B and B1 (SNRPB) promoter, a SPC24/NDC80 kinetochorecomplex component (SPC24) promoter, a transforming acidic coiled-coilcontaining protein 3 (TACC3) promoter, a TBC1 domain family member 31(TBC1D31) promoter, a thymidine kinase 1 (TK1) promoter, a zinc fingerprotein 695 (ZNF695) promoter, an aurora kinase A (AURKA) promoter, aBLM RecQ like helicase (BLM) promoter, a chromosome 17 open readingframe 53 (C17orf53) promoter, a chromobox 3 (CBX30) promoter, a cyclinB1 (CCNB1) promoter, a cyclin E1 (CCNE1) promoter, a cyclin F (CCNF), acell division cycle 20 (CDC20) promoter, a cell division cycle 45(CDC45) promoter, a cell division cycle associated 5 (CDCA5) promoter, acyclin dependent kinase inhibitor 3 (CDKN3) promoter, a cadherin EGF LAGseven-pass G-type receptor 3 (CELSR3) promoter, a centromere protein A(CENPA) promoter, a centrosomal protein 72 (CEP72) promoter, a CDC28protein kinase regulatory subunit 2 (CKS2) promoter, a collagen type Xalpha 1 chain (COL10A1) promoter, a chromosome segregation 1 like(CSE1L) promoter, a DBF4 zinc finger promoter, a GINS complex subunit 1(GINS1) promoter, a G protein-coupled receptor 19 (GPR19) promoter, akinesin family member 18A (KIF18A) promoter, a kinesin family member 4A(KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, aminichromosome maintenance 10 replication initiation factor (MCM10)promoter, a minichromosome maintenance complex component 2 (MCM2)promoter, a minichromosome maintenance complex component 7 (MCM7)promoter, a MRG domain binding protein (MRGBP) promoter, amethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, a non-SMCcondensin I complex subunit H (NCAPH) promoter, a NDC80, kinetochorecomplex component (NDC80) promoter, a nudix hydrolase 1 (NUDT1)promoter, a ribonuclease H2 subunit A (RNASEH2A) promoter, a RuvB likeAAA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancerantigen NY-BR-85 (SGOL1) promoter, a SHC binding and spindle associated1 (SHCBP1) promoter, a small nuclear ribonucleoprotein polypeptide G(SNRPG) promoter, a timeless circadian regulator promoter, a thyroidhormone receptor interactor 13 (TRIP13) promoter, a trophinin associatedprotein (TROAP) promoter, a ubiquitin conjugating enzyme E2 C (UBE2C)promoter, a WD repeat and HMG-box DNA binding protein 1 (WDHD1)promoter, an alpha fetoprotein (AFP) promoter, a fragment thereof, orany combination thereof.
 2. The method of claim 1, wherein said relativeratio is a concentration ratio.
 3. The method of claim 1, wherein saidbiomarker is detected in a biological sample from said subject.
 4. Themethod of claim 3, wherein said biological sample is a bodily fluid fromsaid subject.
 5. The method of claim 4, wherein said biological sampleis a blood or blood-based sample from said subject.
 6. (canceled) 7.(canceled)
 8. The method of claim 1, wherein said subject is a mammal.9. (canceled)
 10. (canceled)
 11. The method of claim 3, wherein saidbiological sample is measured in situ within a human or animal body. 12.The method of any one of claim 1, wherein said composition comprises anucleic acid vector.
 13. The method of claim 12, wherein said vector isselected from the group consisting of nanoplasmids, plasmids,minicircles, recombinant viral vectors, or CELiDs.
 14. (canceled) 15.(canceled)
 16. The method of any one of claim 12, wherein saidcomposition comprises a promoter operably linked to a nucleotidesequence encoding the biomarker.
 17. The method of claim 16, whereinsaid promoter drives expression in a plurality of different types ofdiseased cells in said subject.
 18. The method of claim 17, wherein saidpromoter drives expression of said biomarker in said diseased pluralityof different types of diseased cells preferentially over expression ofsaid biomarker in said non-diseased cells in said subject. 19.(canceled)
 20. The method of claim 16, wherein said biomarker isselected from the group consisting of MRI reporter, a PET reporter, aSPECT reporter, a photoacoustic reporter, a bioluminescent reporter, afluorescent reporter, chemiluminescent reporter, luminescence reporter,colorimetric reporter, a quantifiable nucleic acid biomarker, and anycombination thereof.
 21. The method of claim 20, wherein thequantifiable nucleic acid biomarker is an engineered miRNA.
 22. Themethod of claim 20, wherein detection of said biomarker determines alocation of the diseased cell.
 23. The method of claim 1, wherein saidbiomarker is detectable in a bodily sample of said subject, bynon-invasive imaging or combinations thereof.
 24. The method of claim23, wherein said biomarker is detected using a blood-based assay. 25.The method of any one of claim 1, wherein said composition furthercomprises a transfection agent.
 26. The method of claim 25, wherein saidtransfection agent is a linear or branched polyethylenimine,nanoparticle, lipophilic particle, solid nanoparticle, peptide, micelle,dendrimer, polymeric composition, hydrogel, synthetic or naturallyderived exosome, virus-like particles, or any combination thereof. 27.The method of claim 1, wherein said composition further comprises apharmaceutically acceptable carrier. 28.-44. (canceled)
 45. The methodof claim 1, wherein said promoter comprises a plurality of promotersselected from the group consisting of promoters of BIRC6, CXCR4, ABCC4,AGR2, AICDA, B3GNT3, CDH3, CEACAM5, CENPF, CEP55, CLDN3, CLDN4,COLL11A1, COL1A1, CST1, DTL, FAM111B, FOXA1, KIF20A, MMP1, MMP12, MMP13,MSLN, MUC1, PLA2G2D, RGS13, SCGB2A1, TOP2A, UBD, UBE2C, USH1C, VTCN1,surfactant protein A, c-erbB-2, cyclooxygenase-2, human telomerasereverse transcriptase, TTF-1, UBE2T, CHEK1, E2F1, FOXM1, MYBL2, NEK2,CCNB1, CDC20, CDKN3, COL10A1, KIF4A, MCM10, MCM7, TRIP13, TROAP, UBE2C,and AFP, or any combination thereof.
 46. The method of claim 45, whereinsaid promoter comprises a plurality of promoters selected from the groupconsisting of promoters of ABCC4, B3GNT3, CEACAM5, CEP55, CLDN4, KIF20A,UBE2C, CDC20, CDKN3, COL10A1, MCM10, TRIP13, TROAP, UBE2C, and AFP, orany combination thereof.